U.S. patent application number 11/754235 was filed with the patent office on 2008-11-06 for production of isoprenoids.
Invention is credited to Jack Newman, Christopher John Paddon, Rika Regentin, Neil Stephen Renninger, Keith Kinkead Rolling.
Application Number | 20080274523 11/754235 |
Document ID | / |
Family ID | 38779388 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274523 |
Kind Code |
A1 |
Renninger; Neil Stephen ; et
al. |
November 6, 2008 |
PRODUCTION OF ISOPRENOIDS
Abstract
The present invention provides methods for a robust production
of isoprenoids via one or more biosynthetic pathways. The invention
also provides nucleic acids, enzymes, expression vectors, and
genetically modified host cells for carrying out the subject
methods. The invention also provides fermentation methods for high
productivity of isoprenoids from genetically modified host
cells.
Inventors: |
Renninger; Neil Stephen;
(Oakland, CA) ; Newman; Jack; (Berkeley, CA)
; Rolling; Keith Kinkead; (Oakland, CA) ;
Regentin; Rika; (Hayward, CA) ; Paddon; Christopher
John; (Pacifica, CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
38779388 |
Appl. No.: |
11/754235 |
Filed: |
May 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60808989 |
May 26, 2006 |
|
|
|
60870592 |
Dec 18, 2006 |
|
|
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Current U.S.
Class: |
435/157 ;
435/155; 435/166; 435/167 |
Current CPC
Class: |
C12P 7/04 20130101; C12P
5/007 20130101; C12N 15/52 20130101; C12P 7/02 20130101; C12P 7/00
20130101; C12P 5/026 20130101 |
Class at
Publication: |
435/157 ;
435/166; 435/167; 435/155 |
International
Class: |
C12P 5/00 20060101
C12P005/00; C12P 5/02 20060101 C12P005/02; C12P 7/04 20060101
C12P007/04; C12P 7/02 20060101 C12P007/02 |
Claims
1. A method for producing an isoprenoid comprising: (a) obtaining a
plurality of host cells that comprise an enzymatic pathway for
making isopentenyl pyrophosphate and wherein all of the pathway
enzymes are under control of at least one heterologous
transcriptional regulator; and (b) culturing the host cells in a
medium under conditions that are suboptimal as compared to
conditions that would provide for a maximum specific growth rate
for the host cells.
2. The method of claim 1 wherein the pathway is the mevalonate
pathway.
3. The method of claim 1 wherein the pathway is the DXP
pathway.
4. The method of claim 1 wherein the at least one heterologous
transcriptional regulator is inducible.
5. The method of claim 1 wherein the pathway enzymes are under
control of a single transcriptional regulator.
6. The method of claim 1 wherein the pathway enzymes are under
control of a multiple transcriptional regulator.
7. The method of claim 1 wherein the host cells are
prokaryotes.
8. The method of claim 7 wherein the host cells are E. coli.
9. The method of claim 1 wherein the host cells are eukaryotes.
10. The method of claim 1 wherein the host cells are fungi.
11. The method of claim 9 wherein the host cells are S.
cerevisiae.
12. The method of claim 2 wherein the pathway comprises a nucleic
acid sequence encoding a mevalonate pathway enzyme from a
prokaryote having an endogenous mevalonate pathway.
13. The method of claim 12 wherein the prokaryote is of the genus
selected from Enterococcus, Pseudomonas, and Staphylococcus.
14. The method of claim 13 wherein the nucleic acid sequence is
selected from acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA
reductase, and mevalonate kinase.
15. The method of claim 12 wherein the nucleic acid sequence
encodes a Class II HMG reductase.
16. The method of claim 1 wherein the host cells are cultured in
the medium wherein nutrient or temperature or both are maintained
at a level below that which would provide for the maximum specific
growth rate for the host cells.
17. The method of claim 1 wherein the temperature of the medium is
at least about 2-20.degree. C. below that which would provide for
the maximum specific growth rate.
18. The method of claim 17 wherein the host cells are cultured at a
temperature at least about 5.degree. C. below that which would
provide for the maximum specific growth rate.
19. The method of claim 17 wherein the temperature of the medium is
at least about 10.degree. C. below that which would provide for the
maximum specific growth rate.
20. The method of claim 1 wherein the medium comprises a carbon
source and the amount of carbon source will provide for about 90%
or less of the maximum specific growth rate.
21. The method of claim 20 wherein the amount of the carbon source
will provide for about 75% or less of the maximum specific growth
rate.
22. The method of claim 20 wherein the amount of the carbon source
will provide for about 50% or less of the maximum specific growth
rate.
23. The method of claim 20 wherein the amount of the carbon source
will provide for about 25% or less of the maximum specific growth
rate.
24. The method of claim 20 wherein the amount of the carbon source
will provide for about 75%-110% of the maximum specific growth
rate.
25. The method of claim 1 wherein the medium comprises a nitrogen
source present in an amount below that which would provide for
about 90% or less of the maximum specific rate.
26. The method of claim 25 wherein the amount of the nitrogen
source will provide for about 75% or less of the maximum specific
growth rate.
27. The method of claim 25 wherein the amount of the nitrogen
source will provide for 50% or less of the maximum specific growth
rate.
28. The method of claim 25 wherein the amount of the nitrogen
source will provide for about 25% or less of the maximum specific
growth rate.
29. The method of claim 25 wherein the amount of the nitrogen
source will provide for about 75%-10% of the maximum specific
growth rate.
30. The method of claim 1 wherein the isoprenoid is produced in an
amount greater than about 10 grams per liter of medium.
31. The method of claim 1 wherein the isoprenoid is produced in an
amount greater than about 50 mg per gram of dry cell weight.
32. The method of any one of claims 30 or 31 where the amount of
isoprenoid is produced in less than about 150 hours.
33. The method of any one of claims 30 or 31 where the amount of
isoprenoid is produced in less than about 96 hours.
34. The method of any one of claims 30 or 31 where the amount of
isoprenoid is produced in less than about 72 hours.
35. The method of claim 1 wherein the host cells are cultured at a
temperature below that which would provide for the maximum specific
growth rate, and wherein the host cells are cultured in the medium
in which carbon source is maintained at a level below that which
would provide for the maximum specific growth rate.
36. The method of claim 1 wherein the isoprenoid is selected from
the group consisting of a hemiterpene, monoterpene, diterpene,
triterpene, tetraterpene, and polyterpene.
37. The method of claim 1 wherein the isoprenoid is not a
carotenoid.
38. The method of claim 1 wherein the isoprenoid is a
C.sub.5-C.sub.20 isoprenoid.
39. The method of claim 1 wherein the isoprenoid is selected from
the group consisting of abietadiene, amorphadiene, carene,
.alpha.-farnesene, .beta.-farnesene, farnesol, geraniol,
geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol,
ocimene, patchoulol, .beta.-pinene, sabinene, .gamma.-terpinene,
terpinolene and valencene.
40. A method for producing an isoprenoid comprising: (a) obtaining
a plurality of host cells that comprise a nucleic acid sequence
encoding a mevalonate pathway enzyme from a prokaryote having an
endogenous mevalonate pathway; and (b) culturing the host cells in
a medium under conditions that are suboptimal as compared to
conditions that would provide for a maximum specific growth rate
for the host cells.
41. A host cell capable of producing an isoprenoid in an amount
greater than about 10 grams per liter of medium when cultured in a
medium comprising a carbon source.
42. A host cell capable of producing an isoprenoid in an amount
greater than about 50 mg per gram of dry cell weight when cultured
in a medium comprising a carbon source.
43. The method of any one of claims 40 or 41 wherein the amount of
isoprenoid is produced in less than about 150 hours.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional
Application Nos. 60/808,989, filed May 26, 2006 and 60/870,592
filed on Dec. 18, 2006, which are incorporated herein by reference
in their entirety.
BACKGROUND OF THE INVENTION
[0002] Isoprenoids are ubiquitous in nature. They comprise a
diverse family of over 40,000 individual products, many of which
are vital to living organisms. Isoprenoids serve to maintain
cellular fluidity, electron transport, and other metabolic
functions. A vast number of natural and synthetic isoprenoids are
useful as pharmaceuticals, cosmetics, perfumes, pigments and
colorants, fungicides, antiseptics, nutraceuticals, and fine
chemical intermediates.
[0003] An isoprenoid product is typically composed of repeating
five carbon isopentenyl diphosphate (IPP) units, although irregular
isoprenoids and polyterpenes have been reported. In nature,
isoprenoids are synthesized by consecutive condensations of their
precursor IPP and its isomer dimethylallyl pyrophosphate (DMAPP).
Two pathways for these precursors are known. Eukaryotes, with the
exception of plants, generally use the mevalonate-dependent (MEV)
pathway to convert acetyl coenzyme A (acetyl-CoA) to IPP, which is
subsequently isomerized to DMAPP. Prokaryotes, with some
exceptions, typically employ only the mevalonate-independent or
deoxyxylulose-5-phosphate (DXP) pathway to produce IPP and DMAPP.
Plants use both the MEV pathway and the DXP pathway. See Rohmer et
al. (1993) Biochem. J. 295:517-524; Lange et al. (2000) Proc. Natl.
Acad. Sci. USA 97(24):13172-13177; Rohdich et al. (2002) Proc.
Natl. Acad. Sci. USA 99:1158-1163.
[0004] Traditionally, isoprenoids have been manufactured by
extraction from natural sources such as plants, microbes, and
animals. However, the yield by way of extraction is usually very
low due to a number of profound limitations. First, most
isoprenoids accumulate in nature in only small amounts. Second, the
source organisms in general are not amenable to the large-scale
cultivation that is necessary to produce commercially viable
quantities of a desired isoprenoid. Third, the requirement of
certain toxic solvents for isoprenoid extraction necessitates
special handling and disposal procedures, and thus complicating the
commercial production of isoprenoids.
[0005] The elucidation of the MEV and DXP metabolic pathways has
made biosynthetic production of isoprenoids feasible. For instance,
microbes have been engineered to overexpress a part of or the
entire mevalonate pathway for production of an isoprenoid named
amorpha-4,11-diene (U.S. Pat. Nos. 7,172,886 and 7,192,751) Other
efforts have focused on balancing the pool of
glyceraldehyde-3-phosphate and pyruvate, or on increasing the
expression of 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and IPP
isomerase (idi). See Farmer et al. (2001) Biotechnol. Prog.
17:57-61; Kajiwara et al. (1997) Biochem. J. 324:421-426; and Kim
et al. (2001) Biotechnol. Bioeng. 72:408-415.
[0006] Nevertheless, given the very large quantities of isoprenoid
products needed for many commercial applications, there remains a
need for expression systems and fermentation procedures that
produce even more isoprenoids than available with current
technologies. Optimal redirection of microbial metabolism toward
isoprenoid production requires that the introduced biosynthetic
pathway is properly engineered both to funnel carbon to isoprenoid
production efficiently and to prevent build up of toxic levels of
metabolic intermediates over a sustained period of time. The
present invention addresses this need and provides related
advantages as well.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions and methods for
a robust production of isoprenoids by the use of isopentenyl
pyrophosphate pathway enzymes that are under the control of at
least one heterologous regulator or fermentation conditions, either
alone or in combination. Non-limiting examples of suitable
isoprenoids include: hemiterpenes (derived from 1 isoprene unit)
such as isoprene; monoterpenes (derived from 2 isoprene units) such
as myrcene; sesquiterpenes (derived from 3 isoprene units) such as
amorpha-4,11-diene; diterpenes (derived from four isoprene units)
such as taxadiene; triterpenes (derived from 6 isoprene units) such
as squalene; tetraterpenes (derived from 8 isoprenoids) such as
.beta.-carotene; and polyterpenes (derived from more than 8
isoprene units) such as polyisoprene.
[0008] In one aspect, a method of producing an isoprenoid involves
the steps of (a) obtaining a plurality of host cells that comprise
an enzymatic pathway for making isopentenyl pyrophosphate wherein
the all of the pathway enzymes are under control of at least one
heterologous transcriptional regulator; and (b) culturing the host
cells in a medium under conditions that are suboptimal as compared
to conditions that would provide for a maximum specific growth rate
for the host cells. In some embodiments, the pathway is the
mevalonate pathway. In other embodiments, the pathway is the DXP
pathway. In other embodiments, the at least one heterologous
transcriptional regulatory sequence is inducible. In other
embodiments, the pathway enzymes are under control of a single
transcriptional regulator. In other embodiments, the pathway
enzymes are under control of multiple heterologous transcriptional
regulators.
[0009] In some embodiments, the pathway comprises a nucleic acid
sequence encoding a mevalonate pathway enzyme from a prokaryote
having an endogenous mevalonate pathway. Exemplary prokaryotes
having an endogenous mevalonate pathway include but are not limited
to the genus Enterococcus, the genus Pseudomonas, and the genus
Staphylococcus. In one embodiment, the mevalonate pathway enzyme is
selected from acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA
reductase, and mevalonate kinase. In another embodiment, the
heterologous nucleic acid sequence encodes a Class II HMG-CoA
reductase.
[0010] In another embodiment, the host cells are cultured in a
medium wherein the nutrient and/or temperature level is maintained
at a level below that which would provide for the maximum specific
growth rate for the host cells. In another embodiment, the host
cells are cultured in a medium where the carbon source is
maintained at a level to provide for less than about 90%, 75%, 50%,
25%, 10%, or anywhere between 90% and 10% of the maximum specific
growth rate. In another embodiment, the host cells are cultured in
a medium where the nitrogen source is maintained at a level to
provide for less than about 90%, 75%, 50%, 25%, 10%, or anywhere
between 90% and 10% of the maximum specific growth rate. In another
embodiment, the host cells are cultured in a medium where the
temperature is maintained at a level to provide for less than about
90%, 75%, 50%, 25%, 10% or anywhere between 90% and 10% of the
maximum specific growth rate. In another embodiment, the medium
temperature is maintained at least about 2.degree. C., 4.degree.
C., 5.degree. C., 6.degree. C., 8.degree. C., 10.degree. C.,
15.degree. C., or 20.degree. C. below the temperature that would
provide for the maximum specific growth rate.
[0011] In yet another embodiment, a method of producing an
isoprenoid or isoprenoid precursor comprises the steps of (i)
performing a fermentation reaction comprising a fermentation medium
and a plurality of genetically modified host cells that produce the
isoprenoid under conditions such that (a) the fermentation medium
is kept at a temperature lower than that which would provide for a
maximum specific growth rate of said host cells; (b) the
fermentation medium comprises a carbon source present in an amount
that is lower than that which would provide for a maximum specific
growth rate of the host cells; and/or (c) the fermentation medium
comprises a nitrogen source present in an amount that is lower than
that which would provide for a maximum specific growth rate of the
host cells; (ii) recovering the isoprenoid produced under one or
more conditions set forth in (a) through (c). In one aspect, the
isoprenoid is produced under at least two of the conditions set
forth in (a) through (c). In another aspect, the isoprenoid is
produced under all of the conditions set forth in (a) through
(c).
INCORPORATION BY REFERENCE
[0012] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A is a schematic representation of the mevalonate
("MEV") pathway for the production of isopentenyl pyrophosphate
("IPP").
[0014] FIG. 1B is a schematic representation of the
1-deoxy-D-xylulose 5-diphosphate ("DXP") pathway for the production
of isopentenyl pyrophosphate ("IPP") and dimethylallyl
pyrophosphate ("DMAPP"). Dxs is 1-deoxy-D-xylulose-5-phosphate
synthase; Dxr is 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(also known as IspC); IspD is
4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspE is
4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspF is
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG is
1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG); and
ispH is isopentenyl/dimethylallyl diphosphate synthase.
[0015] FIG. 2 is a schematic representation of the conversion of
isopentenyl pyrophosphate ("IPP") and dimethylallyl pyrophosphate
("DMAPP") to geranyl pyrophosphate ("GPP"), farnesyl pyrophosphate
("FPP"), and geranylgeranyl pyrophosphate ("GGPP"), and the
synthesis of various isoprenoids.
[0016] FIG. 3 shows a map of expression plasmid pMBIS-gpps.
[0017] FIG. 4 shows a map of expression plasmid pAM408.
[0018] FIG. 5 shows a map of expression plasmid pAM424.
[0019] FIG. 6 shows a map of expression plasmids pTrc99A-ADS,
pTrc99A-FSA, pTrc99A-LLS, pTrc99A-LMS, pTrc99A-GTS, pTrc99A-APS,
pTrc99A-BPS, pTrc99A-PHS, pTrc99A-TS, pTrc99A-CS, pTrc99A-SS, and
pAM373.
[0020] FIGS. 7A-C are schematics for the construction of plasmids
pAM489-pAM498 and for pAM328.
[0021] FIG. 8 shows the higher specific activity and increased
stability of the Enterococcus faecalis HMGR-CoA reductase (HMGR)
compared to the Saccharomyces cerevisiae truncated HMG-CoA
reductase (tHMGR).
[0022] FIG. 9 shows the relationship between dry cell weight
("DCW") per liter and OD.sub.600.
[0023] FIGS. 10A-B show the increased volumetric and specific
amorpha-4,11-diene productivity of host strains carrying the
Staphylococcus aureus HMGR and HMGS genes compared to host strains
carrying the Saccharomyces cerevisiae tHMGR and HMGS genes.
[0024] FIG. 11A-B show the effect of lower temperature on
amorpha-4,11-diene productivity of an Escherichia coli host
strain.
[0025] FIGS. 12A-D show the effect of reduced glucose levels on
amorpha-4,11-diene productivity of an Escherichia coli host
strain.
[0026] FIGS. 13A-B show the combined effects of lower temperature
and reduced glucose levels on amorpha-4,11-diene productivity of an
Escherichia coli host strain.
[0027] FIGS. 14A-E and 15A-E show the combined effects of lower
temperature and reduced glucose and nitrogen levels on
amorpha-4,11-diene productivity of an Escherichia coli host
strain.
[0028] FIG. 16 shows production of amorpha-4,11-diene via the DXP
pathway by an Escherichia coli host strain.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Reference is made here to a number of terms that shall be defined
to have the following meanings:
[0030] The term "optional" or "optionally" means that the
subsequently described feature or structure may or may not be
present, or that the subsequently described event or circumstance
may or may not occur, and that the description includes instances
where a particular feature or structure is present and instances
where the feature or structure is absent, or instances where the
event or circumstance occurs and instances where the event or
circumstance does not occur.
[0031] The terms "metabolic pathway" is used herein to refer to a
catabolic pathway or an anabolic pathway. Anabolic pathways involve
constructing a larger molecule from smaller molecules, a process
requiring energy. Catabolic pathways involve breaking down of
larger molecules, often releasing energy.
[0032] The term "mevalonate pathway" or "MEV pathway" is used
herein to refer to the biosynthetic pathway that converts
acetyl-CoA to IPP. The MEV pathway is illustrated schematically in
FIG. 1A.
[0033] The term "deoxyxylulose 5-phosphate pathway" or "DXP
pathway" is used herein to refer to the pathway that converts
glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. The DXP
pathway is illustrated schematically in FIG. 1B.
[0034] The word "pyrophosphate" is used interchangeably herein with
"diphosphate".
[0035] The terms "expression vector" or "vector" refer to a nucleic
acid that transduces, transforms, or infects a host cell, thereby
causing the cell to produce nucleic acids and/or proteins other
than those that are native to the cell, or to express nucleic acids
and/or proteins in a manner that is not native to the cell.
[0036] The term "endogenous" refers to a substance or process that
occurs naturally, e.g., in a non-recombinant host cell.
[0037] The terms "enzymatic pathway for making isopentenyl
pyrophosphate" refers to any pathway capable of producing isopentyl
pyrophosphate, including, without limitation, either the mevalonate
pathway or the DXP pathway.
[0038] The term "nucleic acid" refers to a polymeric form of
nucleotides of any length, either ribonucleotides or
deoxynucleotides. Thus, this term includes, but is not limited to,
single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,
DNA-RNA hybrids, or a polymer comprising purine and pyrimidine
bases or other natural, chemically, or biochemically modified,
non-natural, or derivatized nucleotide bases.
[0039] The term "operon" is used to refer to two or more contiguous
nucleotide sequences that each encode a gene product such as a RNA
or a protein, and the expression of which are coordinately
regulated by one or more controlling elements (for example, a
promoter).
[0040] The term "gene product" refers to RNA encoded by DNA (or
vice versa) or protein that is encoded by an RNA or DNA, where a
gene will typically comprise one or more nucleotide sequences that
encode a protein, and may also include introns and other non-coding
nucleotide sequences.
[0041] The term "protein" refers to a polymeric form of amino acids
of any length, which can include coded and non-coded amino acids,
chemically or biochemically modified or derivatized amino acids,
and polypeptides having modified peptide backbones.
[0042] The term "heterologous nucleic acid" as used herein refers
to a nucleic acid wherein at least one of the following is true:
(a) the nucleic acid is foreign ("exogenous") to (that is, not
naturally found in) a given host cell; (b) the nucleic acid
comprises a nucleotide sequence that is naturally found in (that
is, is "endogenous to") a given host cell, but the nucleotide
sequence is produced in an unnatural (for example, greater than
expected or greater than naturally found) amount in the cell; (c)
the nucleic acid comprises a nucleotide sequence that differs in
sequence from an endogenous nucleotide sequence, but the nucleotide
sequence encodes the same protein (having the same or substantially
the same amino acid sequence) and is produced in an unnatural (for
example, greater than expected or greater than naturally found)
amount in the cell; or (d) the nucleic acid comprises two or more
nucleotide sequences that are not found in the same relationship to
each other in nature (for example, the nucleic acid is
recombinant).
[0043] A "transgene" refers to a gene that is exogenously
introduced into a host cell. It can comprise an endogenous or
exogenous, or heterologous nucleic acid.
[0044] The term "recombinant host" (also referred to as a
"genetically modified host cell" or "genetically modified host
microorganism") denotes a host cell that comprises a heterologous
nucleic acid of the invention.
[0045] The term "exogenous nucleic acid" refers to a nucleic acid
that is exogenously introduced into a host cell, and hence is not
normally or naturally found in and/or produced by a given cell in
nature.
[0046] The term "regulatory element" refers to transcriptional and
translational control sequences, such as promoters, enhancers,
polyadenylation signals, terminators, protein degradation signals,
and the like, that provide for and/or regulate expression of a
coding sequence and/or production of an encoded polypeptide in a
host cell.
[0047] The term "transformation" refers to a permanent or transient
genetic change induced in a cell following introduction of new
nucleic acid. Genetic change ("modification") can be accomplished
either by incorporation of the new DNA into the genome of the host
cell, or by transient or stable maintenance of the new DNA as an
episomal element. In eukaryotic cells, a permanent genetic change
is generally achieved by introduction of the DNA into the genome of
the cell. In prokaryotic cells, a permanent genetic change can be
introduced into the chromosome or via extrachromosomal elements
such as plasmids and expression vectors, which may contain one or
more selectable markers to aid in their maintenance in the
recombinant host cell.
[0048] The term "operably linked" refers to a juxtaposition wherein
the components so described are in a relationship permitting them
to function in their intended manner. For instance, a promoter is
operably linked to a nucleotide sequence if the promoter affects
the transcription or expression of the nucleotide sequence.
[0049] The term "host cell" and "host microorganism" are used
interchangeably herein to refer to any archae, bacterial, or
eukaryotic living cell into which a heterologous nucleic acid can
be or has been inserted. The term also relates to the progeny of
the original cell, which may not necessarily be completely
identical in morphology or in genomic or total DNA complement to
the original parent, due to natural, accidental, or deliberate
mutation.
[0050] The term "synthetic" as used in reference to nucleic acids
means the annealing of chemically synthesized oligonucleotide
building blocks to form gene segments, which are then enzymatically
assembled to construct the entire gene. Synthesis of nucleic acids
via "chemical means" means that the component nucleotides were
assembled in vitro.
[0051] The term "natural" as applied to a nucleic acid, a cell, or
an organism, refers to a nucleic acid, cell, or organism that is
found in nature. For example, a polypeptide or polynucleotide
sequence that is present in a non-pathological (un-diseased)
organism that can be isolated from a source in nature and that has
not been intentionally modified by a human in the laboratory is
natural.
[0052] The term "naturally occurring" as applied to a nucleic acid,
an enzyme, a cell, or an organism, refers to a nucleic acid,
enzyme, cell, or organism that is found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an
organism that can be isolated from a source in nature and that has
not been intentionally modified by a human in the laboratory is
naturally occurring.
[0053] The term "biologically active fragment" as applied to a
protein, polypeptide or enzyme refers to functional portion(s) of
the proteins or polypeptide or enzyme. Functionally equivalents may
have variant amino acid sequences may arise, e.g., as a consequence
of codon redundancy and functional equivalency which are known to
occur naturally within nucleic acid sequences and the proteins thus
encoded. Functionally equivalent proteins or peptides may
alternatively be constructed via the application of recombinant DNA
technology, in which changes in the protein structure may be
engineered, based on considerations of the properties of the amino
acids being exchanged.
[0054] The terms "isoprenoid", "isoprenoid compound", "isoprenoid
product", "terpene", "terpene compound", "terpenoid", and
"terpenoid compound" are used interchangeably herein. They refer to
compounds that are capable of being derived from IPP.
[0055] The singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an expression vector" includes a single
expression vector as well as a plurality of expression vectors, and
reference to "the host cell" includes reference to one or more host
cells, and so forth. It is further noted that the claims may be
drafted to exclude any optional element. As such, this statement is
intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative"
limitation.
[0056] Unless otherwise indicated, this invention is not limited to
particular sequences, expression vectors, enzymes, host
microorganisms, or processes, as such may vary in accordance with
the understanding of those of ordinary skill in the arts to which
this invention pertains in view of the teaching herein. Terminology
used herein is for purposes of describing particular embodiments
only and is not intended to be limiting.
Host Cells
[0057] Any suitable host cell may be used in the practice of the
present invention. In one embodiment, the host cell is a
genetically modified host microorganism in which nucleic acid
molecules have been inserted, deleted or modified (i.e., mutated;
e.g., by insertion, deletion, substitution, and/or inversion of
nucleotides), to either produce the desired isoprenoid compound or
isoprenoid derivative, or effect an increased yield of the desired
isoprenoid compound or isoprenoid derivative. In another
embodiment, the host cell is capable of being grown in liquid
growth medium. In contrast, a "control cell" is an alternative
subject or sample used in an experiment for comparison purpose, and
is typically a parental cell that does not contain the
modification(s) made to a corresponding host cell.
[0058] Illustrative examples of suitable host cells include any
archae, prokaryotic, or eukaryotic cell. Examples of an archae cell
include, but are not limited to those belonging to the genera:
Aeropyrum, Archaeglobus, Halobacterium, Methanococcus,
Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma.
Illustrative examples of archae strains include but are not limited
to: Aeropyrum pernix, Archaeoglobus fulgidus, Methanococcus
jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus
abyssi, Pyrococcus horikoshii, Thermoplasma acidophilum,
Thermoplasma volcanium.
[0059] Examples of a procaryotic cell include, but are not limited
to those belonging to the genera: Agrobacterium, Alicyclobacillus,
Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus,
Brevibacterium, Chromatium, Clostridium, Corynebacterium,
Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus,
Mesorhizobium, Methylobacterium, Microbacterium, Phormidium,
Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum,
Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella,
Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas.
[0060] Illustrative examples of prokaryotic bacterial strains
include but are not limited to: Bacillus subtilis, Bacillus
amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium
immariophilum, Clostridium beigerinckii, Enterobacter sakazakii,
Escherichia coli, Lactococcus lactis, Mesorhizobium loti,
Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica,
Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum
rubrum, Salmonella enterica, Salmonella typhi, Salmonella
typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella
sonnei, Staphylococcus aureus, and the like.
[0061] In general, if a bacterial host cell is used, a
non-pathogenic strain is preferred. Illustrative examples of
non-pathogenic strains include but are not limited to: Bacillus
subtilis, Escherichia coli, Lactibacillus acidophilus,
Lactobacillus helveticus, Pseudomonas aeruginosa, Pseudomonas
mevalonii, Pseudomonas pudita, Rhodobacter sphaeroides, Rodobacter
capsulatus, Rhodospirillum rubrum, and the like.
[0062] Examples of eukaryotic cells include but are not limited to
fungal cells. Examples of fungal cell include, but are not limited
to those belonging to the genera: Aspergillus, Candida,
Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium,
Neurospora, Penicillium, Pichia, Saccharomyces, Trichoderma and
Xanthophyllomyces (formerly Phaffia).
[0063] Illustrative examples of eukaryotic strains include but are
not limited to: Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Candida albicans, Chrysosporium lucknowense,
Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis,
Neurospora crassa, Pichia angusta, Pichia finlandica, Pichia
kodamae, Pichia membranaefaciens, Pichia methanolica, Pichia
opuntiae, Pichia pastoris, Pichia pijperi, Pichia quercuum, Pichia
salictaria, Pichia thermotolerans, Pichia trehalophila, Pichia
stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens,
Streptomyces aureus, Saccaromyces bayanus, Saccaromyces boulardi,
Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomyces
griseochromogenes, Streptomyces griseus, Streptomyces lividans,
Streptomyces olivogriseus, Streptomyces rameus, Streptomyces
tanashiensis, Streptomyces vinaceus, Trichoderma reesei and
Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma).
[0064] In general, if a eukaryotic cell is used, a non-pathogenic
strain is preferred. Illustrative examples of non-pathogenic
strains include but are not limited to: Fusarium graminearum,
Fusarium venenatum, Pichia pastoris, Saccaromyces boulardi, and
Saccaromyces cerevisiae.
[0065] In addition, certain strains have been designated by the
Food and Drug Administration as GRAS or Generally Regarded As Safe.
These strains include: Bacillus subtilis, Lactibacillus
acidophilus, Lactobacillus helveticus, and Saccharomyces
cerevisiae.
IPP Pathways
[0066] The host cells of the present invention comprise or utilize
the MEV pathway, the DXP pathway or both to synthesize IPP and its
isomer, DMAPP. In general, eukaryotes other than plants use the MEV
isoprenoid pathway exclusively to convert acetyl-CoA to IPP, which
is subsequently isomerized to DMAPP. Prokaryotes, with some
exceptions, use the mevalonate-independent or DXP pathway to
produce IPP and DMAPP separately through a branch point. In
general, plants use both the MEV and DXP pathways for IPP
synthesis.
MEV Pathway
[0067] A schematic representation of the MEV pathway is described
in FIG. 1A. In general, the pathway comprises six steps.
[0068] In the first step, two molecules of acetyl-coenzyme A are
enzymatically combined to form acetoacetyl-CoA. An enzyme known to
catalyze this step is, for example, acetyl-CoA thiolase (also known
as acetyl-CoA acetyltransferase). Illustrative examples of
nucleotide sequences include but are not limited to the following
GenBank accession numbers and the organism from which the sequences
derived: (NC.sub.--000913 REGION: 2324131 . . . 2325315;
Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428;
Saccharomyces cerevisiae).
[0069] In the second step of the MEV pathway, acetoacetyl-CoA is
enzymatically condensed with another molecule of acetyl-CoA to form
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to
catalyze this step is, for example, HMG-CoA synthase. Illustrative
examples of nucleotide sequences include but are not limited to:
(NC.sub.--001145. complement 19061 . . . 20536; Saccharomyces
cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882;
Arabidopsis thaliana), (AB037907; Kitasatospora griseola),
(BT007302; Homo sapiens), and (NC.sub.--002758, Locus tag SAV2546,
GeneID 1122571; Staphylococcus aureus).
[0070] In the third step, HMG-CoA is enzymatically converted to
mevalonate. An enzyme known to catalyze this step is, for example,
HMG-CoA reductase. Illustrative examples of nucleotide sequences
include but are not limited to: (NM 206548; Drosophila
melanogaster), (NC.sub.--002758, Locus tag SAV2545, GeneID 1122570;
Staphylococcus aureus), (NM.sub.--204485; Gallus gallus),
(AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotiana
attenuata), (AB037907; Kitasatospora griseola), (AX128213,
providing the sequence encoding a truncated HMGR; Saccharomyces
cerevisiae), and (NC.sub.--001145: complement (115734 . . . 118898;
Saccharomyces cerevisiae).
[0071] In the fourth step, mevalonate is enzymatically
phosphorylated to form mevalonate 5-phosphate. An enzyme known to
catalyze this step is, for example, mevalonate kinase. Illustrative
examples of nucleotide sequences include but are not limited to:
(L77688; Arabidopsis thaliana), and (X55875; Saccharomyces
cerevisiae).
[0072] In the fifth step, a second phosphate group is enzymatically
added to mevalonate 5-phosphate to form mevalonate 5-pyrophosphate.
An enzyme known to catalyze this step is, for example,
phosphomevalonate kinase. Illustrative examples of nucleotide
sequences include but are not limited to: (AF429385; Hevea
brasiliensis), (NM.sub.--006556; Homo sapiens), and
(NC.sub.--001145. complement 712315 . . . 713670; Saccharomyces
cerevisiae).
[0073] In the sixth step, mevalonate 5-pyrophosphate is
enzymatically converted into IPP. An enzyme known to catalyze this
step is, for example, mevalonate pyrophosphate decarboxylase.
Illustrative examples of nucleotide sequences include but are not
limited to: (X97557; Saccharomyces cerevisiae), (AF290095;
Enterococcus faecium), and (U49260; Homo sapiens).
[0074] If IPP is to be converted to DMAPP, then a seventh step is
required. An enzyme known to catalyze this step is, for example,
IPP isomerase. Illustrative examples of nucleotide sequences
include but are not limited to: (NC.sub.--000913, 3031087 . . .
3031635; Escherichia coli), and (AF082326; Haematococcus
pluvialis). If the conversion to DMAPP is required, an increased
expression of IPP isomerase ensures that the conversion of IPP into
DMAPP does not represent a rate-limiting step in the overall
pathway.
DXP Pathway
[0075] A schematic representation of the DXP pathway is described
in FIG. 1B. In general, the DXP pathway comprises seven steps. In
the first step, pyruvate is condensed with D-glyceraldehyde
3-phosphate to make 1-deoxy-D-xylulose-5-phosphate. An enzyme known
to catalyze this step is, for example,
1-deoxy-D-xylulose-5-phosphate synthase. Illustrative examples of
nucleotide sequences include but are not limited to: (AF035440;
Escherichia coli), (NC.sub.--002947, locus tag PP0527; Pseudomonas
putida KT2440), (CP000026, locus tag SPA2301; Salmonella enterica
Paratyphi, see ATCC 9150), (NC.sub.--007493, locus tag
RSP.sub.--0254; Rhodobacter sphaeroides 2.4.1), (NC.sub.--005296,
locus tag RPA0952; Rhodopseudomonas palustris CGA009),
(NC.sub.--004556, locus tag PD1293; Xylella fastidiosa Temecula1),
and (NC.sub.--003076, locus tag AT5G11380; Arabidopsis
thaliana).
[0076] In the second step, 1-deoxy-D-xylulose-5-phosphate is
converted to 2C-methyl-D-erythritol-4-phosphate. An enzyme known to
catalyze this step is, for example, 1-deoxy-D-xylulose-5-phosphate
reductoisomerase. Illustrative examples of nucleotide sequences
include but are not limited to: (AB013300; Escherichia coli),
(AF148852; Arabidopsis thaliana), (NC.sub.--002947, locus tag
PP1597; Pseudomonas putida KT2440), (AL939124, locus tag SCO5694;
Streptomyces coelicolor A3(2)), (NC.sub.--007493, locus tag
RSP.sub.--2709; Rhodobacter sphaeroides 2.4.1), and
(NC.sub.--007492, locus tag Pfl.sub.--1107; Pseudomonas fluorescens
PfO-1).
[0077] In the third step, 2C-methyl-D-erythritol-4-phosphate is
converted to 4-diphosphocytidyl-2C-methyl-D-erythritol. An enzyme
known to catalyze this step is, for example,
4-diphosphocytidyl-2C-methyl-D-erythritol synthase. Illustrative
examples of nucleotide sequences include but are not limited to:
(AF230736; Escherichia coli), (NC.sub.--007493, locus_tag
RSP.sub.--2835; Rhodobacter sphaeroides 2.4.1), (NC.sub.--003071,
locus_tag AT2G02500; Arabidopsis thaliana), and (NC.sub.--002947,
locus_tag PP1614; Pseudomonas putida KT2440).
[0078] In the fourth step,
4-diphosphocytidyl-2C-methyl-D-erythritol is converted to
4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. An enzyme
known to catalyze this step is, for example,
4-diphosphocytidyl-2C-methyl-D-erythritol kinase. Illustrative
examples of nucleotide sequences include but are not limited to:
(AF216300; Escherichia coli) and (NC.sub.--007493, locus_tag
RSP.sub.--1779; Rhodobacter sphaeroides 2.4.1).
[0079] In the fifth step,
4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate is converted
to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. An enzyme known to
catalyze this step is, for example, 2C-methyl-D-erythritol
2,4-cyclodiphosphate synthase. Illustrative examples of nucleotide
sequences include but are not limited to: (AF230738; Escherichia
coli), (NC.sub.--007493, locus_tag RSP.sub.--6071; Rhodobacter
sphaeroides 2.4.1), and (NC.sub.--002947, locus_tag PP1618;
Pseudomonas putida KT2440).
[0080] In the sixth step, 2C-methyl-D-erythritol
2,4-cyclodiphosphate is converted to
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzyme known to
catalyze this step is, for example,
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase.
Illustrative examples of nucleotide sequences include but are not
limited to: (AY033515; Escherichia coli), (NC.sub.--002947,
locus_tag PP0853; Pseudomonas putida KT2440), and (NC.sub.--007493,
locus_tag RSP.sub.--2982; Rhodobacter sphaeroides 2.4.1).
[0081] In the seventh step,
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate is converted into
either IPP or its isomer, DMAPP. An enzyme known to catalyze this
step is, for example, isopentyl/dimethylallyl diphosphate synthase.
Illustrative examples of nucleotide sequences include but are not
limited to: (AY062212; Escherichia coli) and (NC.sub.--002947,
locus_tag PP0606; Pseudomonas putida KT2440).
[0082] In some embodiments, "cross talk" (or interference) between
the host cell's own metabolic processes and those processes
involved with the production of IPP as provided by the present
invention are minimized or eliminated entirely. For example, cross
talk is minimized or eliminated entirely when the host
microorganism relies exclusively on the DXP pathway for
synthesizing IPP, and a MEV pathway is introduced to provide
additional IPP. Such a host organisms would not be equipped to
alter the expression of the MEV pathway enzymes or process the
intermediates associated with the MEV pathway. Organisms that rely
exclusively or predominately on the DXP pathway include, for
example, Escherichia coli.
[0083] In some embodiments, the host cell produces IPP via the MEV
pathway, either exclusively or in combination with the DXP pathway.
In other embodiments, a host's DXP pathway is functionally disabled
so that the host cell produces IPP exclusively through a
heterologously introduced MEV pathway. The DXP pathway can be
functionally disabled by disabling gene expression or inactivating
the function of one or more of the DXP pathway enzymes.
C.sub.5 Compounds
[0084] C.sub.5 compounds of the invention generally are derived
from IPP or DMAPP. These compounds are also known as hemiterpenes
because they are derived from a single isoprene unit (IPP or
DMAPP).
[0085] Isoprene
[0086] Isoprene, whose structure is
##STR00001##
[0087] is found in many plants. Isoprene is made from IPP by
isoprene synthase. Illustrative examples of suitable nucleotide
sequences include but are not limited to: (AB198190; Populus alba)
and (AJ294819; Polulus alba.times.Polulus tremula).
C.sub.10 Compounds
[0088] C.sub.10 compounds of the invention generally derived from
geranyl pyrophosphate (GPP) which is made by the condensation of
IPP with DMAPP. An enzyme known to catalyze this step is, for
example, geranyl pyrophosphate synthase. These C.sub.10 compounds
are also known as monoterpenes because they are derived from two
isoprene units.
[0089] FIG. 2 shows schematically how IPP and DMAPP can produce
GPP, which can be further processed to a monoterpene.
[0090] Illustrative examples of nucleotide sequences for geranyl
pyrophosphate synthase include but are not limited to: (AF513111;
Abies grandis), (AF513112; Abies grandis), (AF513113; Abies
grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum
majus), (Y17376; Arabidopsis thaliana), (AE016877, Locus AP11092;
Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis),
(AY534745; Clarkia breweri), (AY953508; Ips pini), (DQ286930;
Lycopersicon esculentum), (AF182828; Mentha.times.piperita),
(AF182827; Mentha.times.piperita), (MP1249453;
Mentha.times.piperitax), (PZE431697, Locus CAD24425; Paracoccus
zeaxanthinifaciens), (AY866498; Picrorhiza kurrooa), (AY351862;
Vitis vinifera), and (AF203881, Locus AAF12843; Zymomonas
mobilis).
[0091] GPP is then subsequently converted to a variety of C.sub.10
compounds. Illustrative examples of C.sub.10 compounds include but
are not limited:
[0092] Carene
[0093] Carene, whose structure is
##STR00002##
[0094] is found in the resin of many trees, particularly pine
trees. Carene is made from GPP from carene synthase. Illustrative
examples of suitable nucleotide sequences include but are not
limited to: (AF461-460, REGION 43 . . . 1926; Picea abies) and
(AF527416, REGION: 78 . . . 1871; Salvia stenophylla).
[0095] Geraniol
[0096] Geraniol (also known as rhodnol), whose structure is
##STR00003##
[0097] is the main component of oil-of-rose and palmarosa oil. It
also occurs in geranium, lemon, and citronella. Geraniol is made
from GPP by geraniol synthase. Illustrative examples of suitable
nucleotide sequences include but are not limited to: (AJ457070;
Cinnamomum tenuipilum), (AY362553; Ocimum basilicum), (DQ234300;
Perilla frutescens strain 1864), (DQ234299; Perilla citriodora
strain 1861), (DQ234298; Perilla citriodora strain 4935), and
(DQ088667; Perilla citriodora)
[0098] Linalool
[0099] Linalool, whose structure is
##STR00004##
[0100] is found in many flowers and spice plants such as coriander
seeds. Linalool is made from GPP by linalool synthase. Illustrative
examples of a suitable nucleotide sequence include but are not
limited to: (AF497-485; Arabidopsis thaliana), (AC002294, Locus
AAB71482; Arabidopsis thaliana), (AY059757; Arabidopsis thaliana),
(NM.sub.--104793; Arabidopsis thaliana), (AF154124; Artemisia
annua), (AF067603; Clarkia breweri), (AF067602; Clarkia concinna),
(AF067601; Clarkia breweri), (U58314; Clarkia breweri), (AY840091;
Lycopersicon esculentum), (DQ263741; Lavandula angustifolia),
(AY083653; Mentha citrate), (AY693647; Ocimum basilicum),
(XM.sub.--463918; Oryza sativa), (AP004078, Locus BAD07605; Oryza
sativa), (XM.sub.--463918, Locus XP.sub.--463918; Oryza sativa),
(AY917193; Perilla citriodora), (AF271259; Perilla frutescens),
(AY473623; Picea abies), (DQ195274; Picea sitchensis), and
(AF444798; Perilla frutescens var. crispa cultivar No. 79).
[0101] Limonene
[0102] Limonene, whose structure is
##STR00005##
[0103] is found in the rind of citrus fruits and peppermint.
Limonene is made from GPP by limonene synthase. Illustrative
examples of suitable nucleotide sequences include but are not
limited to: (+)-limonene synthases (AF514287, REGION: 47 . . .
1867; Citrus limon) and (AY055214, REGION: 48 . . . 1889; Agastache
rugosa) and (-)-limonene synthases (DQ195275, REGION: 1 . . . 1905;
Picea sitchensis), (AF006193, REGION: 73 . . . 1986; Abies
grandis), and (MHC4SLSP, REGION: 29 . . . 1828; Mentha
spicata).
[0104] Myrcene
[0105] Myrcene, whose structure is
##STR00006##
[0106] is found in the essential oil in many plants including bay,
verbena, and myrcia from which it gets its name. Myrcene is made
from GPP by myrcene synthase. Illustrative examples of suitable
nucleotide sequences include but are not limited to: (U87908; Abies
grandis), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum
majus), (NM.sub.--127982; Arabidopsis thaliana TPS10),
(NM.sub.--113485; Arabidopsis thaliana ATTPS-CIN),
(NM.sub.--113483; Arabidopsis thaliana ATTPS-CIN), (AF271259;
Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea
abies), and (AJ304839; Quercus ilex).
[0107] Ocimene
[0108] .alpha.- and .beta.-Ocimene, whose structures are
##STR00007##
[0109] are found in a variety of plants and fruits including Ocimum
basilicum and is made from GPP by ocimene synthase. Illustrative
examples of suitable nucleotide sequences include but are not
limited to: (AY195607; Antirrhinum majus), (AY195609; Antirrhinum
majus), (AY195608; Antirrhinum majus), (AK221024; Arabidopsis
thaliana), (NM.sub.--113485; Arabidopsis thaliana ATTPS-CIN),
(NM.sub.--113483; Arabidopsis thaliana ATTPS-CIN),
(NM.sub.--117775; Arabidopsis thaliana ATTPS03),
(NM.sub.--001036574; Arabidopsis thaliana ATTPS03),
(NM.sub.--127982; Arabidopsis thaliana TPS10), (AB110642; Citrus
unshiu CitMTSL4), and (AY575970; Lotus corniculatus var.
japonicus).
[0110] .alpha.-Pinene
[0111] .alpha.-Pinene, whose structure is
##STR00008##
[0112] is found in pine trees and eucalyptus. .alpha.-Pinene is
made from GPP by .alpha.-pinene synthase. Illustrative examples of
suitable nucleotide sequences include but are not limited to:
(+).alpha.-pinene synthase (AF543530, REGION: 1 . . . 1887; Pinus
taeda), (-).alpha.-pinene synthase (AF543527, REGION: 32 . . .
1921; Pinus taeda), and (+)/(-).alpha.-pinene synthase (AGU87909,
REGION: 6111892; Abies grandis).
[0113] .beta.-Pinene
[0114] .beta.-Pinene, whose structure is
##STR00009##
[0115] is found in pine trees, rosemary, parsley, dill, basil, and
rose. .beta.-Pinene is made from GPP by .beta.-pinene synthase.
Illustrative examples of suitable nucleotide sequences include but
are not limited to: (-).beta.-pinene synthases (AF276072, REGION: 1
. . . 1749; Artemisia annua) and (AF514288, REGION: 26 . . . 1834;
Citrus limon).
[0116] Sabinene
[0117] Sabinene, whose structure is
##STR00010##
[0118] is found in black pepper, carrot seed, sage, and tea trees.
Sabinene is made from GPP by sabinene synthase. An illustrative
example of a suitable nucleotide sequence includes but is not
limited to AF051901, REGION: 26 . . . 1798 from Salvia
officinalis.
[0119] .gamma.-Terpinene
[0120] .gamma.-Terpinene, whose structure is
##STR00011##
[0121] is a constituent of the essential oil from citrus fruits.
Biochemically, .gamma.-terpinene is made from GPP by a
.gamma.-terpinene synthase. Illustrative examples of suitable
nucleotide sequences include: (AF514286, REGION: 30 . . . 1832 from
Citrus limon) and (AB110640, REGION 1 . . . 1803 from Citrus
unshiu).
[0122] Terpinolene
[0123] Terpinolene, whose structure is
##STR00012##
[0124] is found in black currant, cypress, guava, lychee, papaya,
pine, and tea. Terpinolene is made from GPP by terpinolene
synthase. An illustrative example of a suitable nucleotide sequence
includes but is not limited to AY906866, REGION: 10 . . . 1887 from
Pseudotsuga menziesii.
C.sub.15 Compounds
[0125] C.sub.15 compounds of the invention generally derive from
farnesyl pyrophosphate (FPP) which is made by the condensation of
two molecules of IPP with one molecule of DMAPP. An enzyme known to
catalyze this step is, for example, farnesyl pyrophosphate
synthase. These C.sub.15 compounds are also known as sesquiterpenes
because they are derived from three isoprene units.
[0126] FIG. 2 shows schematically how IPP and DMAPP can be combined
to produce FPP, which can be further processed to a
sesquiterpene.
[0127] Illustrative examples of nucleotide sequences for farnesyl
pyrophosphate synthase include but are not limited to: (ATU80605;
Arabidopsis thaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376;
Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli
K-12), (AE009951, Locus AAL95523; Fusobacterium nucleatum subsp.
nucleatum ATCC 25586), (GFFPPSGEN; Gibberella fujikuroi),
(CP000009, Locus AAW60034; Gluconobacter oxydans 621H), (AF019892;
Helianthus annuus), (HUMFAPS; Homo sapiens), (KLPFPSQCR;
Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771;
Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora
crassa), (PAFPS1; Parthenium argentatum), (PAFPS2; Parthenium
argentatum), (RATFAPS; Rattus norvegicus), (YSCFPP; Saccharomyces
cerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, Locus
AAT87386; Streptococcus pyogenes), (CP000017, Locus AAZ51849;
Streptococcus pyogenes), (NC.sub.--008022, Locus YP.sub.--598856;
Streptococcus pyogenes MGAS10270), (NC.sub.--008023, Locus
YP.sub.--600845; Streptococcus pyogenes MGAS2096),
(NC.sub.--008024, Locus YP.sub.--602832; Streptococcus pyogenes
MGAS10750), and (MZEFPS; Zea mays).
[0128] Alternatively, FPP can also be made by adding IPP to GPP.
Illustrative examples of nucleotide sequences encoding for an
enzyme capable of this reaction include but are not limited to:
(AE000657, Locus AAC06913; Aquifex aeolicus VF5), (NM.sub.--202836;
Arabidopsis thaliana), (D84432, Locus BAA12575; Bacillus subtilis),
(U12678, Locus AAC28894; Bradyrhizobium japonicum USDA 110),
(BACFDPS; Geobacillus stearothermophilus), (NC.sub.--002940, Locus
NP.sub.--873754; Haemophilus ducreyi 35000HP), (L42023, Locus
AAC23087; Haemophilus influenzae Rd KW20), (J05262; Homo sapiens),
(YP.sub.--395294; Lactobacillus sakei subsp. sakei 23K),
(NC.sub.--005823, Locus YP.sub.--000273; Leptospira interrogans
serovar Copenhageni str. Fiocruz L1-130), (AB003187; Micrococcus
luteus), (NC.sub.--002946, Locus YP.sub.--208768; Neisseria
gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp.
NGR234), (J05091; Saccharomyces cerevisae), (CP000031, Locus
AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, Locus AAK99890;
Streptococcus pneumoniae R6), and (NC.sub.--004556, Locus NP
779706; Xylella fastidiosa Temecula1).
[0129] FPP is then subsequently converted to a variety of C.sub.15
compounds. Illustrative examples of C.sub.15 compounds include but
are not limited to:
[0130] Amorphadiene
[0131] Amorphadiene, whose structure is
##STR00013##
[0132] is a precursor to artemisinin which is made by Artemisia
anna. Amorphadiene is made from FPP by amorphadiene synthase. An
illustrative example of a suitable nucleotide sequence is SEQ ID
NO. 37 of U.S. Pat. No. 7,192,751.
[0133] .alpha.-Farnesene
[0134] .alpha.-Farnesene, whose structure is
##STR00014##
[0135] is found in various biological sources including but not
limited to the Dufour's gland in ants and in the coating of apple
and pear peels. .alpha.-Farnesene is made from FPP by
.alpha.-farnesene synthase. Illustrative examples of suitable
nucleotide sequences include but are not limited to DQ309034 from
Pyrus communis cultivar d'Anjou (pear; gene name AFS1) and AY182241
from Malus domestica (apple; gene AFS1). Pechouus et al., Planta
219(1):84-94 (2004).
[0136] .beta.-Farnesene
[0137] .beta.-Farnesene, whose structure is
##STR00015##
[0138] is found in various biological sources including but not
limited to aphids and essential oils such as from peppermint. In
some plants such as wild potato, .beta.-farnesene is synthesized as
a natural insect repellent. .beta.-Farnesene is made from FPP by
.beta.-farnesene synthase. Illustrative examples of suitable
nucleotide sequences include but is not limited to GenBank
accession number AF024615 from Mentha.times.piperita (peppermint;
gene Tspa11), and AY835398 from Artemisia annua. Picaud et al.,
Phytochemistry 66(9): 961-967 (2005).
[0139] Farnesol
[0140] Farnesol, whose structure is
##STR00016##
[0141] is found in various biological sources including insects and
essential oils such as from cintronella, neroli, cyclamen, lemon
grass, tuberose, and rose. Farnesol is made from FPP by a
hydroxylase such as farnesol synthase. Illustrative examples of
suitable nucleotide sequences include but are not limited to
GenBank accession number AF529266 from Zea mays and YDR481c from
Saccharomyces cerevisiae (gene Pho8). Song, L., Applied
Biochemistry and Biotechnology 128:149-158 (2006).
[0142] Nerolidol
[0143] Nerolidol, whose structure is
##STR00017##
[0144] is also known as peruviol, and is found in various
biological sources including as essential oils such as from neroli,
ginger, jasmine, lavender, tea tree, and lemon grass. Nerolidol is
made from FPP by a hydroxylase such as nerolidol synthase. An
illustrative example of a suitable nucleotide sequence includes but
is not limited to AF529266 from Zea mays (maize; gene tps1).
[0145] Patchoulol
[0146] Patchoulol, whose structure is
##STR00018##
[0147] is also known as patchouli alcohol and is a constituent of
the essential oil of Pogostemon patchouli. Patchouliol is made from
FPP by patchouliol synthase. An illustrative example of a suitable
nucleotide sequence includes but is not limited to AY508730 REGION:
1 . . . 1659 from Pogostemon cablin.
[0148] Valencene
[0149] Valencene, whose structure is
##STR00019##
[0150] is one of the main chemical components of the smell and
flavour of oranges and is found in orange peels. Valencene is made
from FPP by nootkatone synthase. Illustrative examples of a
suitable nucleotide sequence includes but is not limited to
AF441124 REGION: 1 . . . 1647 from Citrus sinensis and AY917195
REGION: 1 . . . 1653 from Perilla frutescens.
C.sub.20 Compounds
[0151] C.sub.20 compounds of the invention generally derived from
geranylgeraniol pyrophosphate (GGPP) which is made by the
condensation of three molecules of IPP with one molecule of DMAPP.
An enzyme known to catalyze this step is, for example,
geranylgeranyl pyrophosphate synthase. These C.sub.20 compounds are
also known as diterpenes because they are derived from four
isoprene units.
[0152] FIG. 2 shows schematically how IPP and DMAPP can be combined
to produce GGPP, which can be further processed to a diterpene, or
can be further processed to produce a carotenoid.
[0153] Illustrative examples of nucleotide sequences for
geranylgeranyl pyrophosphate synthase include but are not limited
to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis
thaliana), (NM.sub.--119845; Arabidopsis thaliana),
(NZ_AAJM01000380, Locus ZP.sub.--00743052; Bacillus thuringiensis
serovar israelensis, ATCC 35646 sq1563), (CRGGPPS; Catharanthus
roseus), (NZ_AABF02000074, Locus ZP.sub.--00144509; Fusobacterium
nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN; Gibberella
fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Hevea
brasiliensis), (AB017971; Homo sapiens), (MC1276129; Mucor
circinelloides f. lusitanicus), (AB016044; Mus musculus),
(AABX01000298, Locus NCU01427; Neurospora crassa), (NCU20940;
Neurospora crassa), (NZ_AAKL01000008, Locus ZP.sub.--00943566;
Ralstonia solanacearum UW551), (AB118238; Rattus norvegicus),
(SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus
elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus
acidocaldarius), (NC.sub.--007759, Locus YP.sub.--461832;
Syntrophus aciditrophicus SB), and (NC.sub.--006840, Locus
YP.sub.--204095; Vibrio fischeri ES114).
[0154] Alternatively, GGPP can also be made by adding IPP to FPP.
Illustrative examples of nucleotide sequences encoding an enzyme
capable of this reaction include but are not limited to:
(NM.sub.--112315; Arabidopsis thaliana), (ERWCRTE; Pantoea
agglomerans), (D90087, Locus BAA14124; Pantoea ananatis), (X52291,
Locus CAA36538; Rhodobacter capsulatus), (AF195122, Locus AAF24294;
Rhodobacter sphaeroides), and (NC.sub.--004350, Locus
NP.sub.--721015; Streptococcus mutans UA159).
[0155] GGPP is then subsequently converted to a variety of C.sub.20
isoprenoids. Illustrative examples of C.sub.20 compounds include
but are not limited to:
[0156] Geranylgeraniol
[0157] Geranylgeraniol, whose structure is
##STR00020##
[0158] is a constituent of wood oil from Cedrela toona and of
linseed oil. Geranylgeraniol can be made by e.g., adding to the
expression constructs a phosphatase gene after the gene for a GGPP
synthase.
[0159] Abietadiene
[0160] Abietadiene encompasses the following isomers:
##STR00021##
[0161] and is found in trees such as Abies grandis. Abietadiene is
made by abietadiene synthase. An illustrative example of a suitable
nucleotide sequence includes but are not limited to: (U50768; Abies
grandis) and (AY473621; Picea abies).
C.sub.20+ Compounds
[0162] C.sub.20+ compounds are also within the scope of the present
invention. Illustrative examples of such compounds include
sesterterpenes (C.sub.25 compound made from five isoprene units),
triterpenes (C.sub.30 compounds made from six isoprene units), and
tetraterpenes (C.sub.40 compound made from eight isoprene units).
These compounds are made by using similar methods described herein
and substituting or adding nucleotide sequences for the appropriate
synthase(s).
Engineering Pathways
[0163] The present invention utilizes an engineered MEV and/or DXP
pathway to effect the high-level production of isoprenoids in a
host cell. The pathway is typically engineered via recombinant DNA
technology by expressing heterologous sequences encoding enzymes in
at least one of these pathways.
[0164] The subject nucleotide acids can be expressed by a single or
multiple vectors. For example, a single expression vector can
comprise at least two, three, four, five, or all of the
heterologous sequences encoding the entire MEV or DXP pathway
enzymes. While the choice of single or multiple vectors may depend
on the size of the heterologous sequences and the capacity of the
vectors, it will largely dependent on the overall yield of a given
isoprenoid that the vector is able to provide when expressed in a
selected host cell. The subject vectors can stay replicable
episomally, or as an integral part of the host cell genome.
Typically, the latter is preferred for a sustained propagation of
the host cell.
[0165] In certain host cells, the one or more heterologous
sequences encoding the MEV or DXP pathway enzymes may be controlled
by one or more operons. In some instances, a two or three operon
system provides a higher yield of an isoprenoid over a single
operon system.
[0166] Where desired, the subject nucleic acid sequences can be
modified to reflect the codon preference of a selected host cell to
effect a higher expression of such sequences in a host cell. For
example, the subject nucleotide sequences will in some embodiments
be modified for yeast codon preference. See, e.g., Bennetzen and
Hall (1982) J: Biol. Chem. 257(6): 3026-3031. As another
non-limiting example, the nucleotide sequences will in other
embodiments be modified for E. coli codon preference. See, e.g.,
Gouy and Gautier (1982) Nucleic Acids Res. 10(22):7055-7074;
Eyre-Walker (1996) Mol. Biol. Evol. 13(6):864-872. See also
Nakamura et al. (2000) Nucleic Acids Res. 28(1):292. Codon usage
tables for many organisms are available, which can be used as a
reference in designing sequences of the present invention. The use
of prevalent codons of a given host microorganism generally
increases the likelihood of translation, and hence the expression
level of the desired sequences.
[0167] Preparation of the subject nucleic acids can be carried out
by a variety of routine recombinant techniques and synthetic
procedures. Briefly, the subject nucleic acids can be prepared
genomic DNA fragments, cDNAs, and RNAs, all of which can be
extracted directly from a cell or recombinantly produced by various
amplification processes including but not limited to PCR and
rt-PCR.
[0168] Direct chemical synthesis of nucleic acids typically
involves sequential addition of 3'-blocked and 5'-blocked
nucleotide monomers to the terminal 5'-hydroxyl group of a growing
nucleotide polymer chain, wherein each addition is effected by
nucleophilic attack of the terminal 5'-hydroxyl group of the
growing chain on the 3'-position of the added monomer, which is
typically a phosphorus derivative, such as a phosphotriester,
phosphoramidite, or the like. Such methodology is known to those of
ordinary skill in the art and is described in the pertinent texts
and literature (for example, Matteuci et al. (1980) Tet. Lett.
521:719; U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat.
Nos. 5,436,327 and 5,700,637 to Southern et al.).
[0169] The level of transcription of a nucleic acid in a host
microorganism can be increased in a number of ways. For example,
this can be achieved by increasing the copy number of the
nucleotide sequence encoding the enzyme (e.g., by using a higher
copy number expression vector comprising a nucleotide sequence
encoding the enzyme, or by introducing additional copies of a
nucleotide sequence encoding the enzyme into the genome of the host
microorganism, for example, by recA-mediated recombination, use of
"suicide" vectors, recombination using lambda phage recombinase,
and/or insertion via a transposon or transposable element). In
addition, it can be carried out by changing the order of the coding
regions on the polycistronic mRNA of an operon or breaking up an
operon into individual genes, each with its own control elements,
or increasing the strength of the promoter (transcription
initiation or transcription control sequence) to which the enzyme
coding region is operably linked (for example, using a consensus
arabinose- or lactose-inducible promoter in an Escherichia coli
host microorganism in place of a modified lactose-inducible
promoter, such as the one found in pBluescript and the pBBR1MCS
plasmids), or using an inducible promoter and inducing the
inducible-promoter by adding a chemical to a growth medium. The
level of translation of a nucleotide sequence in a host
microorganism can be increased in a number of ways, including, but
not limited to, increasing the stability of the mRNA, modifying the
sequence of the ribosome binding site, modifying the distance or
sequence between the ribosome binding site and the start codon of
the enzyme coding sequence, modifying the entire intercistronic
region located "upstream of" or adjacent to the 5' side of the
start codon of the enzyme coding region, stabilizing the 3'-end of
the mRNA transcript using hairpins and specialized sequences,
modifying the codon usage of enzyme, altering expression of rare
codon tRNAs used in the biosynthesis of the enzyme, and/or
increasing the stability of the enzyme, as, for example, via
mutation of its coding sequence. Determination of preferred codons
and rare codon tRNAs can be based on a sequence analysis of genes
derived from the host microorganism.
[0170] The activity of a MEV, DXP, or prenyltransferase in a host
can be altered in a number of ways, including, but not limited to,
expressing a modified form of the enzyme that exhibits increased
solubility in the host cell, expressing an altered form of the
enzyme that lacks a domain through which the activity of the enzyme
is inhibited, expressing a modified form of the enzyme that has a
higher Kcat or a lower Km for the substrate, or expressing an
altered form of the enzyme that is not affected by feed-back or
feed-forward regulation by another molecule in the pathway. Such
variant enzymes can also be isolated through random mutagenesis of
a broader specificity enzyme, as described below, and a nucleotide
sequence encoding such variant enzyme can be expressed from an
expression vector or from a recombinant gene integrated into the
genome of a host microorganism.
[0171] The subject vector can be constructed to yield a desired
level of copy numbers of the encoded enzyme. In some embodiments,
the subject vectors yield at least 10, between 10 to 20, between
20-50, between 50-100, or even higher than 100 copies of the
HMG-CoA reductase, mevalonate kinase, or both. Low copy number
plasmids generally provide fewer than about 20 plasmid copies per
cell; medium copy number plasmids generally provide from about 20
plasmid copies per cell to about 50 plasmid copies per cell, or
from about 20 plasmid copies per cell to about 80 plasmid copies
per cell; and high copy number plasmids generally provide from
about 80 plasmid copies per cell to about 200 plasmid copies per
cell, or more.
[0172] Suitable low copy expression vectors for Escherichia coli
include, but are not limited to, pACYC184, pBeloBac11, pBR332,
pBAD33, pBBR1MCS and its derivatives, pSC101, SuperCos (cosmid),
and pWE15 (cosmid). Suitable medium copy expression vectors for
Escherichia coli include, but are not limited to pTrc99A, pBAD24,
and vectors containing a ColE1 origin of replication and its
derivatives. Suitable high copy number expression vectors for
Escherichia coli include, but are not limited to, pUC, pBluescript,
pGEM, and pTZ vectors. Suitable low-copy (centromeric) expression
vectors for yeast include, but are not limited to, pRS415 and
pRS416 (Sikorski & Hieter (1989) Genetics 122:19-27). Suitable
high-copy 2 micron expression vectors in yeast include, but are not
limited to, pRS425 and pRS426 (Christainson et al. (1992) Gene
110:119-122). Alternative 2 micron expression vectors include
non-selectable variants of the 2 micron vector (Bruschi &
Ludwig (1988) Curr. Genet. 15:83-90) or intact 2 micron plasmids
bearing an expression cassette (as exemplified in U.S. Pat. Appl.
20050084972) or 2 micron plasmids bearing a defective selection
marker such as LEU2d (Erhanrt et al. (1983) J. Bacteriol. 156 (2):
625-635) or URA3d (Okkels (1996) Annals of the New York Academy of
Sciences 782(1): 202-207).
[0173] Regulatory elements include, for example, promoters and
operators can also be engineered to increase the metabolic flux of
the MEV or DXP pathways by increasing the expression of one or more
genes that play a significant role in determining the overall yield
of an isoprenoid produced. A promoter is a sequence of nucleotides
that initiates and controls the transcription of a nucleic acid
sequence by an RNA polymerase enzyme. An operator is a sequence of
nucleotides adjacent to the promoter that functions to control
transcription of the desired nucleic acid sequence. The operator
contains a protein-binding domain where a specific repressor
protein can bind. In the absence of a suitable repressor protein,
transcription initiates through the promoter. In the presence of a
suitable repressor protein, the repressor protein binds to the
operator and thereby inhibits transcription from the promoter.
Promotors and operators are also referred to as transcriptional
regulators.
[0174] In some embodiments of the present invention, promoters used
in expression vectors are inducible. In other embodiments, the
promoters used in expression vectors are constitutive. In some
embodiments, one or more nucleic acid sequences are operably linked
to an inducible promoter, and one or more other nucleic acid
sequences are operably linked to a constitutive promoter.
[0175] Non-limiting examples of suitable promoters for use in
prokaryotic host cells include a bacteriophage T7 RNA polymerase
promoter; a trp promoter; a lac operon promoter; a hybrid promoter,
for example, a lac/tac hybrid promoter, a tac/trc hybrid promoter,
a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac
promoter, and the like; an araBAD promoter; in vivo regulated
promoters, such as an ssaG promoter or a related promoter (see, for
example, U.S. Patent Publication No. 20040131637), a pagC promoter
(Pulkkinen and Miller, J. Bacteriol. (1991) 173(1):86-93;
Alpuche-Aranda et al. (1992) Proc. Natl. Acad. Sci. USA.
89(21):10079-83), a nirB promoter (Harborne et al. (1992) Mol.
Micro. 6:2805-2813), and the like (see, for example, Dunstan et al.
(1999). Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine
22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892);
a sigma70 promoter, for example, a consensus sigma70 promoter (see,
for example, GenBank Accession Nos. AX798980, AX798961, and
AX798183); a stationary phase promoter, for example, a dps
promoter, an spv promoter, and the like; a promoter derived from
the pathogenicity island SPI-2 (see, for example, WO96/17951); an
actA promoter (see, for example, Shetron-Rama et al. (2002) Infect.
Immun. 70:1087-1096); an rpsM promoter (see, for example, Valdivia
and Falkow (1996) Mol. Microbiol. 22:367 378); a tet promoter (see,
for example, Hillen et al. (1989) In Saenger W. and Heinemann U.
(eds) Topics in Molecular and Structural Biology, Protein-Nucleic
Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an
SP6 promoter (see, for example, Melton et al. (1984) Nucl. Acids
Res. 12:7035-7056); and the like.
[0176] In some embodiment, the total activity of a heterologous MEV
or DXP enzyme that plays a larger role in the overall yield of an
isoprenoid relative to other enzymes in the respective pathways is
increased by expressing the enzyme from a strong promoter. Suitable
strong promoters for Escherichia coli include, but are not limited
to Trc, Tac, T5, T7, and P.sub.Lambda. In another embodiment of the
present invention, the total activity of the one or more MEV
pathway enzymes in a host is increased by expressing the enzyme
from a strong promoter on a high copy number plasmid. Suitable
examples, for Escherichia coli include, but are not limited to
using Trc, Tac, T5, T7, and P.sub.Lambda promoters with pBAD24,
pBAD18, pGEM, pBluescript, pUC, and pTZ vectors.
[0177] Non-limiting examples of suitable promoters for use in
eukaryotic host cells include, but are not limited to, a CMV
immediate early promoter, an HSV thymidine kinase promoter, an
early or late SV40 promoter, LTRs from retroviruses, and a mouse
metallothionein-I promoter.
[0178] Non-limiting examples of suitable constitutive promoters for
use in prokaryotic host cells include a sigma70 promoter (for
example, a consensus sigma70 promoter). Non-limiting examples of
suitable inducible promoters for use in bacterial host cells
include the pL of bacteriophage .lamda.; Plac; Ptrp; Ptac (Ptrp-lac
hybrid promoter); an isopropyl-beta-D44 thiogalactopyranoside
(IPTG)-inducible promoter, for example, a lacZ promoter; a
tetracycline inducible promoter; an arabinose inducible promoter,
for example, PBAD (see, for example, Guzman et al. (1995) J.
Bacteriol. 177:4121-4130); a xylose-inducible promoter, for
example, Pxyl (see, for example, Kim et al. (1996) Gene 181:71-76);
a GAL1 promoter; a tryptophan promoter; a lac promoter; an
alcohol-inducible promoter, for example, a methanol-inducible
promoter, an ethanol-inducible promoter; a raffinose-inducible
promoter; a heat-inducible promoter, for example, heat inducible
lambda PL promoter; a promoter controlled by a heat-sensitive
repressor (for example, CI857-repressed lambda-based expression
vectors; see, for example, Hoffmann et al. (1999) FEMS Microbiol
Lett. 177(2):327-34); and the like.
[0179] Non-limiting examples of suitable constitutive promoters for
use in yeast host cells include an ADH1, an ADH2, a PGK, or a LEU2
promoter. Non-limiting examples of suitable inducible promoters for
use in yeast host cells include, but are not limited to, a
divergent galactose-inducible promoter such as a GAL 1 or a GAL 10
promoter (West at al. (1984) Mol. Cell. Biol. 4(11):2467-2478), or
a CUP1 promoter. Where desired, the subject vector comprise a
promoter that is stronger than a native E. Coli Lac promoter.
[0180] Non-limiting examples of operators for use in bacterial host
cells include a lactose promoter operator (LacI repressor protein
changes conformation when contacted with lactose, thereby
preventing the LacI repressor protein from binding to the
operator), a tryptophan promoter operator (when complexed with
tryptophan, TrpR repressor protein has a conformation that binds
the operator; in the absence of tryptophan, the TrpR repressor
protein has a conformation that does not bind to the operator), and
a tac promoter operator (see, for example, deBoer et al. (1983)
Proc. Natl. Acad. Sci. U.S.A. 80:21-25.).
[0181] The genes in the expression vector typically will also
encode a ribosome binding site to direct translation (that is,
synthesis) of any encoded mRNA gene product. For suitable ribosome
binding sites for use in Escherichia coli, see Shine et al. (1975)
Nature 254:34, and Steitz, in Biological Regulation and
Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p.
349, 1979, Plenum Publishing, N.Y. Insertion of the ribosome
binding site encoding nucleotide sequence 5'-AAAACA-3' upstream of
a coding sequence facilitates efficient translation in a yeast host
microorganism (Looman et al. (1993) Nuc. Ac. Res. 21:4268-4271; Yun
et. al. (1996) Mol. Microbiol. 19:1225-1239).
[0182] Other regulatory elements that may be used in an expression
vector include transcription enhancer elements and transcription
terminators. See, for example, Bitter et al. (1987) Methods in
Enzymology, 153:516-544.
[0183] An expression vector may be suitable for use in particular
types of host microorganisms and not others. One of ordinary skill
in the art, however, can readily determine through routine
experimentation whether a particular expression vector is suited
for a given host microorganism. For example, the expression vector
can be introduced into the host organism, which is then monitored
for viability and expression of any genes contained in the
vector.
[0184] The expression vector may also contain one or more
selectable marker genes that, upon expression, confer one or more
phenotypic traits useful for selecting or otherwise identifying
host cells that carry the expression vector. Non-limiting examples
of suitable selectable markers for eukaryotic cells include
dihydrofolate reductase and neomycin resistance. Non-limiting
examples of suitable selectable markers for prokaryotic cells
include tetracycline, ampicillin, chloramphenicol, carbenicillin,
and kanamycin resistance.
[0185] For production of isoprenoid at an industrial scale, it may
be impractical or too costly to use a selectable marker that
requires the addition of an antibiotic to the fermentation media.
Accordingly, some embodiments of the present invention employ host
cells that do not require the use of an antibiotic resistance
conferring selectable marker to ensure plasmid (expression vector)
maintenance. In these embodiments of the present invention, the
expression vector contains a plasmid maintenance system such as the
60-kb IncP (RK2) plasmid, optionally together with the RK2 plasmid
replication and/or segregation system, to effect plasmid retention
in the absence of antibiotic selection (see, for example, Sia et
al. (1995) J. Bacteriol. 177:2789-97; Pansegrau et al. (1994) J.
Mol. Biol. 239:623-63). A suitable plasmid maintenance system for
this purpose is encoded by the parDE operon of RK2, which codes for
a stable toxin and an unstable antitoxin. The antitoxin can inhibit
the lethal action of the toxin by direct protein-protein
interaction. Cells that lose the expression vector that harbors the
parDE operon are quickly deprived of the unstable antitoxin,
resulting in the stable toxin then causing cell death. The RK2
plasmid replication system is encoded by the trfA gene, which codes
for a DNA replication protein. The RK2 plasmid segregation system
is encoded by the parCBA operon, which codes for proteins that
function to resolve plasmid multimers that may arise from DNA
replication.
[0186] The subject vectors can be introduced into a host cell
stably or transiently by variety of established techniques. For
example, one method involves a calcium chloride treatment wherein
the expression vector is introduced via a calcium precipitate.
Other salts, for example calcium phosphate, may also be used
following a similar procedure. In addition, electroporation (that
is, the application of current to increase the permeability of
cells to nucleic acids) may be used. Other transformation methods
include microinjection, DEAE dextran mediated transformation, and
heat shock in the presence of lithium acetate. Lipid complexes,
liposomes, and dendrimers may also be employed to transfect the
host microorganism.
[0187] Upon transformation, a variety of methods can be practiced
to identify the host cells into which the subject vectors have been
introduced. One exemplary selection method involves subculturing
individual cells to form individual colonies, followed by testing
for expression of the desired gene product. Another method entails
selecting transformed host cells based upon phenotypic traits
conferred through the expression of selectable marker genes
contained within the expression vector. Those of ordinary skill can
identify genetically modified host cells using these or other
methods available in the art.
[0188] The introduction of various pathway sequences of the
invention into a host cell can be confirmed by methods such as PCR,
Southern blot or Northern blot hybridization. For example, nucleic
acids can be prepared from the resultant host cells, and the
specific sequences of interest can be amplified by PCR using
primers specific for the sequences of interest. The amplified
product is subjected to agarose gel electrophoresis, polyacrylamide
gel electrophoresis or capillary electrophoresis, followed by
staining with ethidium bromide, SYBR Green solution or the like, or
detection of DNA with a UV detection. Alternatively, nucleic acid
probes specific for the sequences of interest can be employed in a
hybridization reaction. The expression of a specific gene sequence
can be ascertained by detecting the corresponding mRNA via
reveres-transcription coupled PCR, Northern blot hybridization, or
by immunoassays using antibodies reactive with the encoded gene
product. Exemplary immunoassays include but are not limited to
ELISA, radioimmunoassays, and sandwich immunoassays.
[0189] The enzymatic activity of a given pathway enzyme can be
assayed by a variety of methods known in the art. In general, the
enzymatic activity can be ascertained by the formation of the
product or conversion of a substrate of an enzymatic reaction that
is under investigation. The reaction can take place in vitro or in
vivo. For example, the relative activity of HMG-CoA reductase and
HMG-CoA synthase in a cell can be measured by the steady state
level of HMG-CoA in a cell. HMG-CoA can be extracted by
Tricholoroacetic Acid (TCA), followed by analyzing the extracted
material via Liquid Chromatography/Mass Spectrometry. The activity
of mevalonate kinase can be demonstrated by the formation of
mevalonate 5-phosphate. The relative activity of mevalonate kinase
and HMG-CoA reductase can be measured by the steady state level of
mevalonate, which can be determined by Gas Chromatography/Mass
spectrometry. See e.g., WO05033287, which is incorporated herein by
reference.
[0190] The yield of an isoprenoid via one or more metabolic
pathways disclosed herein can be augmented by inhibiting reactions
that divert intermediates from productive steps towards formation
of the isoprenoid product. Inhibition of the unproductive reactions
can be achieved by reducing the expression and/or activity of
enzymes involved in one or more unproductive reactions. Such
reactions include side reactions of the TCA cycle that lead to
fatty acid biosynthesis, alanine biosynthesis, the aspartate
superpathway, gluconeogenesis, heme biosynthesis, and/or glutamate
biosynthesis, at a level that affects the overall yield of an
isoprenoid production. Additionally, the conversion of acetyl-CoA
to acetate via the action of phosphotransacetylase is another
example of unproductive side reaction. Therefore, where desired,
"knocking out" or "knocking down" the pta gene that encodes
phosphotransacetylase may also be carried in order to increase the
yield of isoprenoid production. Depending on the specific
isoprenoid of interest, one skilled in the art may choose to target
additional unproductive steps. For example, where carotenoid is the
isoprenoid of choice, one may opt to "knock out" or "knock down"
one or more genes selected from the group consisting of gdhA, aceE,
fdhF, yjiD, hnr or yjfP, ackA, appY, aspC, clp, clpP, clpXP, crcB,
csdA, cyaA, evgS, fdhA, fdhD, feoB, fumA, glnE, glxR, gntK, hycI,
lipB, lysU, modA, moeA, nadA, nuoC, nuoK, pflB, pitA, pst, pstC,
pta, p-yjiD, sohA, stpA, yagR, yaiD, ybaS, ycfZ, ydeN, yebB, yedN,
yfcC, ygiP, yibD, yjfP, yjhH, or yliE genes, or any other genes
alone or in combination, the inhibition of which would result in a
higher yield of carotenoid as described in U.S. Patent Application
20060121558, which is incorporated herein by reference.
[0191] A variety of methods are available for knocking out or
knocking down a gene of interest. For example, a reduced gene
expression may be accomplished by deletion, mutation, and/or gene
rearrangement. It can also be carried out with the use of antisense
RNA, siRNA, miRNA, ribozymes, triple stranded DNA, and
transcription and/or translation inhibitors. In addition,
transposons can be employed to disrupt gene expression, for
example, by inserting it between the promoter and the coding
region, or between two adjacent genes to inactivate one or both
genes.
High Yields of Isoprenoid Compounds
[0192] The present invention provides compositions and methods for
a robust production of isoprenoids by the use of isopentenyl
pyrophosphate pathway enzymes that are under the control of at
least one heterologous regulator or fermentation conditions, either
alone or in combination.
[0193] In one aspect, a method of producing an isoprenoid involves
the steps of (a) obtaining a plurality of host cells that comprise
an enzymatic pathway for making isopentenyl pyrophosphate wherein
the all of the pathway enzymes are under control of at least one
heterologous transcriptional regulator; and (b) culturing the host
cells in a medium under conditions that are suboptimal as compared
to conditions that would provide for a maximum specific growth rate
for the host cells. In some embodiments, the pathway is the
mevalonate pathway. In other embodiments, the pathway is the DXP
pathway. In other embodiments, the at least one heterologous
transcriptional regulatory sequence is inducible. In other
embodiments, the pathway enzymes are under control of a single
transcriptional regulator. In other embodiments, the pathway
enzymes are under control of multiple heterologous transcriptional
regulators.
[0194] In some embodiments, the pathway comprises a nucleic acid
sequence encoding a mevalonate pathway enzyme from a prokaryote
having an endogenous mevalonate pathway. Non-limiting examples of
suitable prokaryotes include those from the genera: Actinoplanes,
Archaeoglobus; Bdellovibrio; Borrelia, Chloroflexus; Enterococcus,
Lactobacillus, Listeria; Oceanobacillus; Paracoccus, Pseudomonas,
Staphylococcus, Streptococcus; Streptomyces, Thermoplasma; and
Vibrio. Non-limiting examples of specific strains include:
Archaeoglobus fulgidus; Bdellovibrio bacteriovorus; Borrelia
burgdorferi, Chloroflexus aurantiacus; Enterococcus faecalis;
Enterococcus faecium; Lactobacillus johnsonii; Lactobacillus
plantarum; Lactococcus lactis; Listeria innocua; Listeria
monocytogenes; Oceanobacillus iheyensis, Paracoccus
zeaxanthinifaciens; Pseudomonas mevalonii; Staphylococcus aureus;
Staphylococcus epidermidis; Staphylococcus haemolyticus;
Streptococcus agalactiae; Streptomyces griseolosporeus;
Streptococcus mutans; Streptococcus pneumoniae; Streptococcus
pyogenes; Thermoplasma acidophilum, Thermoplasma volcanium; Vibrio
cholerae; Vibrio parahaemolyticus; and Vibrio vulnificus;
[0195] In another embodiment, the nucleic acid sequence encoding a
mevalonate pathway enzyme is selected from acetyl-CoA thiolase,
HMG-CoA synthase, HMG-CoA reductase, and mevalonate kinase. In
another embodiment, the nucleic acid sequence encoding a mevalonate
pathway enzyme is selected from acetyl-CoA thiolase, HMG-CoA
synthase, HMG-CoA reductase, and mevalonate kinase and is from a
prokaryote belonging to the genus Enterococcus or the genus
Pseudomonas or the genus Staphylococcus. In another embodiment, the
nucleic acid sequence encoding a mevalonate pathway enzyme is
selected from acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA
reductase, and mevalonate kinase and is from Enterococcus faecalis
or from Staphylococcus aureus.
[0196] In another embodiment, nucleic acid sequence encoding a
mevalonate pathway enzyme is a Class II HMG-CoA reductase. HMG-CoA
reductases are generally classified into two classes, which are
distinguishable based on sequence homology and/or enzymatic
properties (see, for example, Hedl, et al., J. Bacteriology,
1927-1932, 2004, and Bochar, et al., Molec. Genet. Metab., 66,
122-127, 1999).
[0197] Class II HMG-CoA reductases can be characterized, in part,
by their low sensitivity to statins, including but not limited to
Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Pravastatin,
Simvastatin, in some embodiments, the Class II HMG-CoA reductase
exhibits a statin inhibition constant greater than about 1
micromolar, 10 micromolar, or 100 micromolar. In other embodiments,
the Class II HMG-CoA reductase has an inhibition constant for
Lovastatin greater than that of the Class I HMG-CoA by a factor of
at least about 10, 100, 1000, or 10,000. In other embodiments, the
Class II HMG-CoA reductase has an inhibition constant for
Lovastatin greater than that of the Class I HMG-CoA isolated from a
Homo sapien by a factor of at least about 10, 100, 1000, or 10,000.
In other embodiments, the Class II HMG-CoA reductase is from a
prokaryote. In other embodiments, the Class II HMG-CoA reductase is
from archae bacteria.
[0198] A prototypical Class II HMG-CoA reductase is derived from
Pseudomonas mevalonii. Also encompassed in the invention are
variant Class II HMG-CoA reductases exhibiting at least about 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or 95% identity
as compared to the amino acid sequence of P. mevalonii HMG-CoA
reductase. Further encompassed in the invention are variants having
less than about 40%, 35%, 30%, 25%, 20%, or less, identity with an
H. Sapiens HMG-CoA reductase. The identities of amino acid
sequences can be determined by the methods described in Bochar, et
al., Molec. Genet. Metab., 66, 122-127, 1999.
[0199] Non-limiting exemplary Class II HMG-CoA reductases include
those derived from HMG-CoA reductases from: Archaeoglobus fulgidus
(NC.sub.--000917), Bdellovibrio bacteriovorus (BX842650); Borrelia
burgdorferi (AE001169); Chloroflexus aurantiacus (AJ299212);
Enterococcus faecalis (AAO81155); Enterococcus faecium (AF290094);
Lactobacillus johnsonii (AE017204); Lactobacillus plantarum;
Lactococcus lactis (AE006387); Listeria innocua (CAC96053);
Listeria monocytogenes (AE017324); Oceanobacillus iheyensis
(NC.sub.--000917); Paracoccus zeaxanthinifaciens (AJ431696);
Pseudomonas mevalonii (M24015); Staphylococcus aureus (AF290086);
Staphylococcus epidermidis (AF290090); Staphylococcus haemolyticus
(AF290088); Streptococcus agalactiae (CAD47046); Streptomyces
griseolosporeus (AB037907); Streptococcus mutans (AAN58647);
Streptococcus pneumoniae (AF290098); Streptococcus pyogenes
(AF290096); Thermoplasma acidophilum (CAC11548), Thermoplasma
volcanium (AL935253); Vibrio cholerae (AAF96622); Vibrio
parahaemolyticus (BAC62311); and Vibrio vulnificus (AA007090).
[0200] The fermentation methods described herein relate to
modulating the specific growth rate of the host cells. Often
represented by the parameter .mu., the specific growth rate
represents the rate of growth of cells per unit of biomass per unit
time. The specific growth rate has the units of reciprocal time
(1/t). The maximum specific growth rate for cells in a culture
medium relates to the effect of substrate concentration on growth
rate. Generally, cells will grow slowly at a low level of the
substrate, and as the level of the substrate in the medium
increases, so does the rate of cell growth. However, the rate of
cell growth does not continue to rise indefinitely, and at high
levels of substrate, a given increase in the amount of substrate
will produce a smaller and smaller increase in the rate of cell
growth. Therefore, the growth rate ultimately reaches a limit,
which is often referred to as the maximum specific growth rate. A
theoretical treatment of the relationship between growth rate in
culture is well known to those skilled in the art, and is referred
to as the Monod equation. See, for example, Pirt, Principles of
Microbe and Cell Cultivation, Wiley, NY, 1975, pages 4-10. In this
theoretical treatment, the maximum specific rate is an asymptotic
limit that is never reached until an infinite level of substrate is
reached. In practice, however, the maximum specific growth rate can
be considered as being obtained when the conditions under
investigation (e.g., a substrate level or temperature) support the
fastest initial growth rate. For instance, in a fed-batch reactor,
the initial condition where the nutrients are supplied in excess is
treated as the conditions for the maximum growth rate. See, for
example, Lee et al. (1996) Trends Biotechnol. 14: 98-105 and Korz
et al. (1995) J Biotechnology 39:59-65. The conditions where a
substrate is added to support the maximum specific growth rate is
also sometimes referred to as unlimited growth. In addition, while
the Monod equation describes the theoretical rate properties for a
substrate that asymptotically approaches the maximum specific rate,
for many substrates, rather than approaching the value as more
substrate is added, a decrease in rate is seen at higher levels of
substrate after a maximum rate is achieved, i.e., the maximum
specific growth rate is achieved followed by a decrease in growth
rate.
[0201] The maximum specific growth rate can also be applied with
respect to temperature as well as to substrates. Generally, an
organism will grow slowly at low temperatures, and will grow at a
faster rate as the temperature increases up to a certain point,
after which, the growth rate will decline. There will be a
temperature at which the growth rate will be at a maximum level,
this is the temperature at which the maximum growth rate is
achieved. We have found that the production of isoprenoids can be
increased by lowering the temperature below the temperature that
supports the maximum specific growth rate.
[0202] The maximum specific growth rate can also be applied with
respect to other additives to the fermentation than substrates. For
instance, with respect to nutrients, vitamins, and minerals, there
can be a low rate at low amounts of these components, the rate will
get higher as the concentration of the component is increased,
then, in some cases, at even higher concentrations of the
components, the rate will decrease. The maximum specific growth
rate is obtained where the concentration of the component supports
the highest rate.
[0203] The maximum specific growth rate for a cell in a medium is
often determined at the initial stages of the fermentation before
inhibition by end product or intermediates, cell crowding, or other
factors contribute to slowing down the rate of growth. For example
the maximum growth rate is often determined during the exponential
phase of growth rather than at the lag phase, deceleration phase,
or the stationary phase. The concept of maximum specific growth
rate can also be applied at later stages of the fermentation by
taking into account the appropriate variables.
[0204] Accordingly, in some embodiments, host cells are cultured
under conditions such that growth is less than about 90% of the
maximum specific growth rate. In other embodiments, the host cells
are cultured under conditions such that growth is less than about
80%, 75%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, 5%, or 1%, or less, of
the maximum specific growth rate.
[0205] In other embodiments, the host cells are cultured at a
medium temperature is at least about 2.degree. C., 4.degree. C.,
5.degree. C., 6.degree. C., 8.degree. C., 10.degree. C., 15.degree.
C., or 20.degree. C. below the temperature that would provide for
the maximum specific growth rate. By lowering the temperature
growth is reduced, which in turn, reduces the formation of toxic
byproducts in the medium and the generation of metabolic heat.
Lowering culture temperature also reduces cellular oxygen demand
which enables higher cell-densities to be obtained.
[0206] The temperature at which at which the maximum specific
growth rate of a host cell can be achieved will depend on the type
of host cell selected. This can be ascertained by growing the host
cells under various temperatures over a defined period of time to
derive the relevant growth curves. The temperature that supports
the maximum specific growth rate can be determined by comparing the
slopes of growth in the respective curves. In the case of E. Coli,
the temperature for maximum specific growth rate is about
37.degree. C. Accordingly, if E. Coli is the host cell to be used
for fermentative production of isoprenoid, the fermentation
temperature is below 37.degree. C. If S. cerevisiae is employed,
the temperature for maximum specific growth rate is about
30.degree. C. Accordingly, if S. cerevisiae is the host cell to be
used for fermentative production of isoprenoid, the fermentation
temperature is below 30.degree. C. Typically a desired temperature
is about 2.degree. C., 4.degree. C., 5.degree. C., 6, 8, 10.degree.
C., 15.degree. C., and 20.degree. C. below the temperature at which
the maximum specific growth rate of the host cell can be
achieved.
[0207] In other embodiments, the host cells are cultured in a
fermentation medium comprises a carbon source present in an amount
that is lower than that which would provide for a maximum specific
growth rate. In certain embodiments, the host cells are cultured in
a medium where the carbon source is maintained at a level to
provide for less than about 90%, 80%, 75%, 60%, 50%, 40%, 30%, 25%,
20%, 10%, 5%, 1%, or less, of the maximum specific growth rate. Any
carbon-containing sources that are digestible by the microorganism
can be used. Non-limiting examples include carbohydrates such as
monosaccharides, oligosaccharides and polysaccharides, organic
acids such as acetic acid, propionic acid; and alcohols such as
ethanol and propanol, and polyols such as glycerol.
[0208] In some embodiments, the carbon sources comprise primarily
monosaccharides or oligosaccharides. In other embodiments, the
carbon source consists essentially of monosaccharides and
disaccharides. In still other embodiments, the carbon source is
essentially free of cellulose.
[0209] Monosaccharides are the simple sugars that serve as building
blocks for carbohydrates. They are classified based on their
backbone of carbon (C) atoms: trioses have three carbon atoms,
tetroses four, pentoses five, hexoses six, and heptoses seven. The
carbon atoms are bonded to hydrogen atoms (--H), hydroxyl groups
(--OH), and carbonyl groups (--C.dbd.O), whose combinations, order,
and configurations allow a large number of stereoisomers to exist.
Pentoses include xylose, found in woody materials; arabinose, found
in gums from conifers; ribose, a component of RNA and several
vitamins, and deoxyribose, a component of DNA. Exemplary hexoses
include glucose, galactose, and fructose. Monosaccharides combine
with each other and other groups to form a variety of
disaccharides, and oligosaccharides. An oligosaccharide is a
saccharide polymer containing a small number (typically three to
ten) of simple sugars. They are generally found either O- or
N-linked to compatible amino acid side chains in proteins or lipid
moieties. A preferred oligosaccharide for use in the present
fermentation reaction is disaccharide, including for example,
sucrose, or trisaccharide such as raffinose.
[0210] Where it is desired to have cellulose, glycan, starch, or
other polysaccharides as the ultimate carbon source, these
polysaccharides can be first converted into monosaccharides and
oligosaccharides by chemical means or by enzymatic methods. For
instance, cellulose can be converted into glucose by the enzyme
cellulase. Accordingly, if polysaccharides such as cellulose found
in the biomass (including e.g., canola, alfalfa, rice, rye,
sorghum, sunflower, wheat, soybean, tobacco, potato, peanut,
cotton, sweet potato, cassava, coffee, coconut, citrus trees,
cocoa, tea, fruits such as, banana, fig, pineapple, guava, mango,
oats, barley, vegetables, ornamentals, or conifers) is used as the
ultimate carbon source, it can be digested by cellulase to generate
simpler sugars for use in conjunction with the fermentation
procedure of the present invention. In certain embodiments, after
the breakdown of the polysaccharide, the monosaccharide and/or
oligosaccharide constitute at least about 50% by weight of the
carbon source as determined at the beginning of the fermentation.
In other embodiments, the monosaccharide and/or oligosaccharide
constitute at least about 80% or even 90% by weight of the carbon
source as determined at the beginning of the fermentation, such
that the fermentation medium is essentially free of cellulose.
[0211] In other embodiments, the host cells are cultured in a
fermentation medium comprises a nitrogen source present in an
amount that is lower than that which would provide for a maximum
specific growth rate. While not being bound by any particular
theory, it is known that changing the levels of components such as
nitrogen that are available to a cell can change the relative flux
through the various chemical pathways within the cell. We have
found that by restricting the level of nitrogen available to the
microorganism, the amount of isoprenoid such as amorphadiene
produced by the microorganism is increased. Exemplary levels of
nitrogen of the present invention include in an amount that would
support about 90%, 80%, 75%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, 5%,
1%, or less, of the maximum specific growth rate.
[0212] The restriction of nitrogen can be implemented in stages. In
some embodiments, nitrogen in the form of ammonia is provided in
the beginning of the fermentation to support initial growth, but
subsequent additions to the fermentation are free of nitrogen, or
are free of nitrogen save for that level of ammonia needed to
maintain the pH of the fermentation at 7 with an ammonia solution.
For the bulk of the fermentation, the level of nitrogen is
maintained at a level which is less than the amount which would
support the maximum specific growth rate. The amounts can be for
example amounts which would support at least about 90%, 80%, 75%,
60%, 50%, 40%, 30%, 25%, 20%, 10%, 5%, or 1%, or less, of the
maximum specific growth rate. A fermentation of the present
invention could have an initial nitrogen level above 10 mM as
measured in the fermentation medium, to support an initial growth,
and a subsequent a nitrogen level below 50 mM, 40 mM, 30 nM, 20 mM,
10 mM, or 4 mM in the fermentation medium.
[0213] Sources of assimilable nitrogen that can be used in a
suitable fermentation reaction mixture include, but are not limited
to, simple nitrogen sources, organic nitrogen sources, and complex
nitrogen sources. Such nitrogen sources include anhydrous ammonia,
ammonium salts of inorganic or organic acids such as ammonium
chloride, ammonium sulfate, ammonium acetate, ammonium phosphate,
other nitrogen-containing compounds and substances of animal,
vegetable, and/or microbial origin. Amino acids can also be used as
the nitrogen source, including leucine, isoleucine or valine, or a
mixture thereof.
[0214] Any known method for providing a substrate to a fermentation
reaction may be used to maintain the substrate level below the
level which would provide for the maximum specific growth rate.
Illustrative examples include the batch method where all the
substrate for the fermentation is added in the beginning of the
fermentation reaction; the continuous feed method; and the variable
feed rate method, where, for instance, an increasing amount of
substrate is provided as the fermentation proceeds in order to
support the increased concentration of cells in the medium.
Combinations of these three methods are often employed. For
instance, it is common to have a certain amount of substrate
present initially in the fermentation, to allow the microorganisms
to deplete this initial amount of substrate, then to subsequently
add substrate either continuously or to add it variably after the
initial amount of substrate is utilized. It is an aspect of this
invention to provide an initial amount of substrate to the cells in
the fermentation medium, which may be present at a relatively high
level, to allow the host cells to substantially use up the initial
substrate, then to subsequently provide substrate to the host cells
at a level that is suboptimal as compared to the amount that would
support the maximum growth rate by either a continuous or variable
feed rate. It will be appreciated by those of skill in the art that
since the cells may be growing at an exponential rate, it can be
advantageous to vary the feed rate at an exponential rate in order
to keep the amount of substrate relative to the level of host cells
constant. Thus in certain embodiments, substrates are added in an
exponentially increasing manner, but yet at a level which is lower
than the level which would provide the maximum specific growth
rate.
[0215] In some embodiments, the fermentation reaction is given a
reduced feed of carbon source relative to the carbon feed which
would provide for the maximum specific growth rate. While not being
bound by a particular theory, it is known that changing the levels
of nutrients available to a cell will change the relative flux
through the various chemical pathways within the cell. For example,
some enzymes are inducible, and will only be active when certain
nutrients are present. We have observed that lowering the carbon
source feed rate to a microorganism can improve the amount of
isoprenoid produced in the fermentation. In practice, the carbon
source can be supplied initially in an amount sufficient to support
an initial growth of the host cells until such initial carbon
source is substantially depleted, after which the carbons source is
added at an exponential rate, but at a rate which is below that
which would support the maximum specific growth of the host cells.
For example, in a method of the present invention, the carbon
source is added in an amount that would support about 90%, 80%,
75%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, or less, of the
maximum specific growth rate.
[0216] In other embodiments, the fermentation reaction is given an
initial bolus of carbon source sufficient to grow at or near the
maximum specific growth rate (unlimited growth) followed by a
reduced feed rate at a level below that required to support the
maximum specific growth rate, for the remainder of the
fermentation. In some cases, the point at which the reduced carbon
source feed rate is implemented is the point at which a
predetermined feed rate is achieved. In certain embodiments, the
microorganism is provided with enough carbon source to grow
exponentially to a feed rate of about 15 g/L/hr, after which the
feed rate is reduced to 5.7 g/L/hr and held constant at that rate
for the remainder of the fermentation.
[0217] In certain embodiments, one or more of the heterologous
mevalonate or DXP pathway enzymes is inducible and induced after
the carbon source feed rate has been reduced to a level below that
required for maximum specific growth. For example, where the
engineered microorganism has an inducible promoter, the
fermentation is first run by adding carbon source to achieve a
exponential growth, but at a level which is below that to support
maximum specific growth, then the carbon source feed rate is
reduced to an even lower level for the remainder of the
fermentation, and the inducer added after the carbon source feed
rate is reduced. In some embodiments, the microorganisms are
induced with isopropylthio-beta-D-galactoside (IPTG) after the
reduced carbon source feed is initiated.
[0218] In other embodiments, the fermentation reaction is performed
in a manner that avoids the build up of toxic substances that
decrease cell growth rates. For example, it is known that when too
much glucose is added to the medium, toxic products such as acetate
can build up in the organism. See, for example, Kortz et al. (1995)
J. Biotechnol 39: 59-65. Thus by providing a high level of carbon
source, at or approaching the amount which would support a maximum
growth rate (unlimited growth), the initial growth of the cells may
be higher, but the growth becomes arrested due to the accumulation
of toxic substances. The level at which the carbon source is added
below the level where the toxic products do not accumulate is
referred to as the critical level or the inhibitory threshold. Thus
in certain embodiments, the fermentation reaction is performed such
that the carbon source is kept below the critical level for the
build up of toxic substances. Those skilled in the art will
appreciate that the critical concentration of substrates will vary
with the strain and the medium which is used.
[0219] An effective fermentation reaction mixture can contain other
compounds such as inorganic salts, vitamins, trace metals, or
growth promoters. Such other compounds can also be present in
carbon, nitrogen or mineral sources in the effective reaction
mixture or can be added specifically to the reaction mixture. One
embodiment of the invention involves providing these compounds at
levels that are suboptimal as compared to that would support the
maximum growth rate of the host cells in order to increase
isoprenoid production.
[0220] The fermentation reaction mixture can also contain a
suitable phosphate source. Such phosphate sources include both
inorganic and organic phosphate sources. Non-limiting examples of
phosphate sources include, but are not limited to, phosphate salts
such as mono or dibasic sodium and potassium phosphates, ammonium
phosphate, polyphosphate, and mixtures thereof. A suitable
fermentation reaction mixture can also include a source of
magnesium. In some embodiments, the magnesium is in the form of a
physiologically acceptable salt, such as magnesium sulfate
heptahydrate, although other magnesium sources in concentrations
that contribute similar amounts of magnesium can be used. Further,
in some instances it may be desirable to allow the fermentation
reaction mixture to become depleted of a magnesium source during
fermentation. In some embodiments, the phosphorous source is
provided in an amount that is suboptimal as compared to that would
support a maximum specific growth rate.
[0221] The fermentation reaction mixture can also include a
biologically acceptable chelating agent, such as the dihydrate of
trisodium citrate and ethylenediaminetetraacetic acid. The
fermentation reaction mixture can also initially include a
biologically acceptable acid or base to maintain the desired pH of
the fermentation reaction mixture. Biologically acceptable acids
include, but are not limited to, hydrochloric acid, sulfuric acid,
nitric acid, phosphoric acid and mixtures thereof. Biologically
acceptable bases include, but are not limited to, ammonium
hydroxide, sodium hydroxide, potassium hydroxide and mixtures
thereof.
[0222] The fermentation reaction mixture can also include a
biologically acceptable calcium source, including, but not limited
to, calcium chloride. The fermentation reaction mixture can also
include sodium chloride. The fermentation reaction mixture can also
include trace metals. Such trace metals can be added to the
fermentation reaction mixture as a stock solution that, for
convenience, can be prepared separately from the rest of the
fermentation reaction mixture. A suitable trace metals solution can
include, but is not limited to sodium selenate; ferrous sulfate;
heptahydrate; cupric sulfate, pentahydrate; zinc sulfate,
heptahydrate; sodium molybdate, dihydrate; cobaltous chloride;
selenium or chromium solution; hexahydrate; and manganous sulfate
monohydrate. Hydrochloric acid may be added to the stock solution
to keep the trace metal salts in solution.
[0223] If a pathway intermediate or a compound that can be
converted to a pathway intermediate is added to the fermentation
medium, the intermediate or compound is typically present in an
excess amount.
[0224] Fermentation can be conducted under anaerobic (deficient in
oxygen) or aerobic (oxygenated) conditions. Under aerobic
conditions, microorganisms can break down sugars to end products
such as CO.sub.2 and H.sub.2O. Under anaerobic conditions, the host
cells utilize an alternative pathway to produce CO.sub.2 and
ethanol. Fermentation can also be used to refer to the bulk growth
of microorganisms on a growth medium where no distinction is made
between aerobic and anaerobic metabolism. In general, aerobic
fermentation is carried out for production of isoprenoids.
[0225] The fermentations of the present invention can be carried
out in a batch, a fed-batch, or a continuous process. A batch
process is typically a closed process where all of the raw
materials are added at the beginning of the fermentation. A
fed-batch process is typically a closed process where the carbon
source and/or other substrates are added in increments throughout
the process. A fed-batch process allows for greater control of the
medium and the growth of the microorganisms. A continuous process
can be considered an open system where medium is continuously added
and product is simultaneously removed. Processes in between these
types can also be used. For instance, in one embodiment, the
fermentation is begun as a fed-batch process, and an organic layer,
such as dodecane is placed in contact with the fermentation medium
while the fermentation process continues. Isoprenoids, which
typically have a higher solubility in the organic medium than in
the aqueous fermentation medium are extracted out of the
fermentation medium into the organic layer. Where the isoprenoids
are produced in excess of the saturation point and form a layer
separable from the medium, then simple separation by way of
draining or sucking the distinct phase layer can be carried out.
This process has characteristics of both a fed-batch process and a
continuous process, because of the removal of product from the
medium and the fermentation progresses. The fed-batch and
continuous processes allow for the control of the addition of
fermentation components during the fermentation process. A
fed-batch, continuous, or combination of these processes is usually
preferred in carrying out the invention. The processes allow for
greater control of the rate of addition of feed and other
fermentation components as a function of time. The removal of
product during fermentation can be beneficial, especially where the
accumulated product leads to inhibition of the production
pathways.
[0226] The amount of microorganism per liter of fermentation, or
the density of microorganism, can be measured by measuring the
weight of microorganism isolated from a given volume of the
fermentation medium. A common measure is the dry weight of cells
per liter of fermentation medium. Another method which can be used
to monitor the fermentation while it is progressing is by a
measurement of the optical density of the medium. A common method
is to measure the optical density at a wavelength of 600 nm,
referred to the OD.sub.600, or the OD. The OD can be correlated to
a the density of a specific type of organism within a specific
medium, but the specific relationship between OD and amount of
microorganism per volume will not generally be applicable across
all types of organisms in all types of media. A calibration curve
can be created by measuring the OD and the dry cell weight over a
range of cell densities. In some cases, these correlations can be
used in different fermentation of the same or similar
microorganisms in the same or similar media.
[0227] In another aspect, the present invention provides a method
comprising the steps of (i) performing a fermentation reaction
comprising a fermentation medium and a plurality of genetically
modified host cells that produce the isoprenoid under conditions
such that (a) the fermentation medium is kept at a temperature
lower than that which would provide for a maximum specific growth
rate of said host cells; (b) the fermentation medium comprises a
carbon source present in an amount that is lower than that which
would provide for a maximum specific growth rate of the host cells;
and/or (c) the fermentation medium comprises a nitrogen source
present in an amount that is lower than that which would provide
for a maximum specific growth rate of the host cells; (ii)
recovering the isoprenoid produced under one or more conditions set
forth in (a) through (c). In one embodiment, the fermentation
reaction is run under condition (a). In another embodiment, the
fermentation reaction is run under conditions (a) and (b). In yet
another embodiment, the fermentation reaction is run under
conditions of (a), (b), and (c), or in any other combinations
thereof.
[0228] Using the methods described herein, the host cells produce
more than about 10 grams of isoprenoid per liter of fermentation
reaction mixture (10 g/L). In other embodiments, more than about 15
g/L, more than about 20 g/L, more than 25 g/L is produced, or more
than about 30 g/L of isoprenoid is produced.
[0229] In another embodiment, the host cells produce more than
about 50 milligrams of isoprenoid per gram of dry host cells (50
milligrams per gram dry cell weight) is produced. In other
embodiments, more than about 100 milligrams per gram dry cell
weight, more than about 150 milligrams per gram dry cell weight,
more than about 200 milligrams per gram dry cell weight, more than
about 250 milligrams per gram dry cell weight, more than about 500
milligrams per gram dry cell weight, more than about 750 milligrams
per gram dry cell weight, or more than about 1000 milligrams per
gram dry cell weight of isoprenoid is produced.
[0230] In other embodiments, the production level, whether it is in
grams per liter or milligrams per gram dry cell weight is achieved
in less than about 150 hours, preferably less than about 96 hours,
or even less than about 72 hours.
[0231] Non-limiting examples of suitable isoprenoids include:
hemiterpenes (derived from 1 isoprene unit) such as isoprene;
monoterpenes (derived from 2 isoprene units) such as myrcene;
sesquiterpenes (derived from 3 isoprene units) such as
amorpha-4,11-diene; diterpenes (derived from four isoprene units)
such as taxadiene; triterpenes (derived from 6 isoprene units)
squalene; tetraterpenes (derived from 8 isoprenoids)
.beta.-carotene; and polyterpenes (derived from more than 8
isoprene units) such as polyisoprene. In some embodiments, the
isoprenoid is not a carotenoid. In other embodiments, the
isoprenoid is a C.sub.5-C.sub.20 isoprenoid.
[0232] Although the invention has been described in conjunction
with specific embodiments thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention.
Other aspects, advantages, and modifications within the scope of
the invention will be apparent to those skilled in the art to which
the invention pertains.
EXAMPLES
[0233] The practice of the present invention can employ, unless
otherwise indicated, conventional techniques of the biosynthetic
industry and the like, which are within the skill of the art. To
the extent such techniques are not described fully herein, one can
find ample reference to them in the scientific literature.
[0234] In the following examples, efforts have been made to ensure
accuracy with respect to numbers used (for example, amounts,
temperature, and so on), but variation and deviation can be
accommodated, and in the event a clerical error in the numbers
reported herein exists, one of ordinary skill in the arts to which
this invention pertains can deduce the correct amount in view of
the remaining disclosure herein. Unless indicated otherwise,
temperature is reported in degrees Celsius, and pressure is at or
near atmospheric pressure at sea level. All reagents, unless
otherwise indicated, were obtained commercially. The following
examples are intended for illustrative purposes only and do not
limit in any way the scope of the present invention.
Example 1
[0235] This example describes methods for making expression
plasmids that encode for enzymes including enzymes of the MEV
pathway from Saccharomyces cerevisiae organized in operons.
[0236] Expression plasmid pMevT was generated by inserting the MevT
operon (SEQ ID NO: 1) into the pBAD33 vector. The MevT operon
encodes the set of MEV pathway enzymes that together transform the
ubiquitous precursor acetyl-CoA to (R)-mevalonate, namely
acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.
The MevT operon was generated by PCR amplifying from Escherichia
coli genomic DNA the coding sequence of the atoB gene (GenBank
accession number NC.sub.--000913 REGION: 2324131 . . . 2325315)
(encodes an acetoacetyl-CoA thiolase), from Saccharomyces
cerevisiae genomic DNA the coding sequence of the ERG13 gene
(GenBank accession number X96617, REGION: 220 . . . 1695) (encodes
a HMG-CoA synthase), and from Saccharomyces cerevisiae genomic DNA
a segment of the coding region of the HMG1 gene (GenBank accession
number M22002, REGION: 1660 . . . 3165) (encodes a truncated
HMG-CoA reductase (tHMGR)). The upstream PCR primer used for the
amplification of the HMG1 gene fragment included an artificial
start codon. The amplified fragments were spliced together using
overlap extensions (SOEing), during which process ribosome binding
sites were introduced after the atoB and the ERG13 coding
sequences. After the addition of 3' A overhangs, the MevT operon
was ligated into the TA cloning vector pCR4 (Invitrogen, Carlsbad,
Calif.), and sequenced to ensure accuracy. The MevT operon was
subsequently ligated into the XmaI PstI restriction enzyme site of
vector pBAD33 (Guzman et al. (1995) J. Bacteriol. 177(14):
4121-4130). To place the operon under the control of the P.sub.Lac
promoter, the araC-P.sub.BADNsiI-XmaI fragment of pBAD33 was
replaced with the NsiI-XmaI fragment of pBBR1MCS, yielding
expression plasmid pMevT (see U.S. Pat. No. 7,192,751).
[0237] Expression plasmid pAM36-MevT66 was generated by inserting
the MevT66 operon into the pAM36 vector. Vector pAM36 was generated
by inserting an oligonucleotide cassette containing
AscI-SfiI-AsiSI-XhoI-PacI-FsIl-PmeI restriction enzyme sites into
the pACYC184 vector (GenBank accession number XO6403), and by
removing the tet resistance gene in pACYC184. The MevT66 operon was
synthetically generated using the nucleotide sequence SEQ ID NO: 1
as a template, which comprises the atoB gene from Escherichia coli
(GenBank accession number NC.sub.--000913 REGION: 2324131 . . .
2325315), the ERG13 gene from Saccharomyces cerevisiae (GenBank
accession number X96617, REGION: 220 . . . 1695), and a truncated
version of the HMG1 gene from Saccharomyces cerevisiae (GenBank
accession number M22002, REGION: 1777 . . . 3285), all three
sequences being codon-optimized for expression in Escherichia coli.
The synthetically generated MevT66 operon was flanked by a 5' EcoRI
restriction enzyme site and a 3' Hind III restriction enzyme site,
and could thus be cloned into compatible restriction enzyme sites
of a cloning vector such as a standard pUC or pACYC origin vector.
From this construct, the MevT66 operon was PCR amplified with
flanking SfiI and AsiSI restriction enzyme sites, the amplified DNA
fragment was digested to completion using SfiI and AsiSI
restriction enzymes, the reaction mixture was resolved by gel
electrophoresis, the approximately 4.2 kb DNA fragment was gel
extracted using a Qiagen gel purification kit (Valencia, Calif.),
and the isolated DNA fragment was ligated into the SfiI AsiSI
restriction enzyme site of the pAM36 vector, yielding expression
plasmid pAM36-MevT66.
[0238] Expression plasmid pAM25 was generated by inserting the
MevT66 operon into the pAM29 vector. Vector pAM29 was created by
assembling the p15A origin of replication and kan resistance gene
from pZS24-MCS1 (Lutz and Bujard (1997) Nucl Acids Res.
25:1203-1210) with an oligonucleotide-generated lacUV5 promoter.
The DNA synthesis construct comprising the MevT66 operon (see
above) was digested to completion using EcoRI and Hind III
restriction enzymes, the reaction mixture was resolved by gel
electrophoresis, the 4.2 kb DNA fragment was gel extracted, and the
isolated DNA fragment was ligated into the EcoRI HindIII
restriction enzyme site of pAM29, yielding expression plasmid
pAM25.
[0239] Expression plasmid pMevB-Cm was generated by inserting the
MevB operon into the pBBR1MCS-1 vector. The MevB operon encodes the
set of enzymes that together convert (R)-mevalonate to IPP, namely
mevalonate kinase, phosphomevalonate kinase, and mevalonate
pyrophosphate carboxylase. The MevB operon was generated by PCR
amplifying from Saccharomyces cerevisiae genomic DNA the coding
sequences of the ERG12 gene (GenBank accession number X55875,
REGION: 580 . . . 1911) (encodes a mevalonate kinase), the ERG8
gene (GenBank accession number Z49939, REGION: 3363 . . . 4718)
(encodes a phosphomevalonate kinase), and the MVD1 gene (GenBank
accession number X97557, REGION: 544 . . . 1734) (encodes a
mevalonate pyrophosphate carboxylase), and by splicing the PCR
fragments together using overlap extensions (SOEing). By choosing
appropriate primer sequences, the stop codons of ERG12 and ERG8
were changed from TAA to TAG during amplification to introduce
ribosome binding sites. After the addition of 3' A overhangs, the
MevB operon was ligated into the TA cloning vector pCR4
(Invitrogen, Carlsbad, Calif.). The MevB operon was excised by
digesting the cloning construct to completion using PstI
restriction enzyme, resolving the reaction mixture by gel
electrophoresis, gel extracting the 4.2 kb DNA fragment, and
ligating the isolated DNA fragment into the PstI restriction enzyme
site of vector pBBR1MCS-1 (Kovach et al., Gene 166(1): 175-176
(1995)), yielding expression plasmid pMevB-Cm.
[0240] Expression plasmid pMBI was generated by inserting the MBI
operon into the pBBR1MCS-3 vector. The MBI operon encodes the same
enzymes as the MevB operon, as well as an isopentenyl
pyrophosphatase isomerase that catalyzes the conversion of IPP to
DMAPP. The MBI operon was generated by PCR amplifying from
Escherichia coli genomic DNA the coding sequence of the idi gene
(GenBank accession number AF119715) using primers that contained an
XmaI restriction enzyme site at their 5' ends, digesting the
amplified DNA fragment to completion using XmaI restriction enzyme,
resolving the reaction mixture by gel electrophoresis, gel
extracting the 0.5 kb fragment, and ligating the isolated DNA
fragment into the XmaI restriction enzyme site of expression
plasmid pMevB-Cm, thereby placing idi at the 3' end of the MevB
operon. The MBI operon was subcloned into the SalI and SacI
restriction enzyme sites of vector pBBR1MCS-3 (Kovach et al., Gene
166(1): 175-176 (1995)), yielding expression plasmid pMBI (see U.S.
Pat. No. 7,192,751).
[0241] Expression plasmid pMBIS was generated by inserting the ispA
gene into pMBI. The ispA gene encodes a farnesyl pyrophosphate
synthase that catalyzes the condensation of two molecules of IPP
with one molecule of DMAPP to make farnesyl pyrophosphate (FPP).
The coding sequence of the ispA gene (GenBank accession number
D00694, REGION: 484 . . . 1383) was PCR amplified from Escherichia
coli genomic DNA using a forward primer with a SacII restriction
enzyme site and a reverse primer with a SacI restriction enzyme
site. The amplified PCR product was digested to completion with
SacII and SacI restriction enzymes, the reaction mixture was
resolved by gel electrophoresis, and the 0.9 kb DNA fragment was
gel extracted. The isolated DNA fragment was ligated into the SacII
SacI restriction enzyme site of pMBI, thereby placing the ispA gene
3' of idi and the MevB operon, and yielding expression plasmid
pMBIS (see U.S. Pat. No. 7,192,751).
[0242] Expression plasmid pMBIS-gpps was derived from expression
plasmid pMBIS by replacing the ispA coding sequence with a
nucleotide sequence encoding a geranyl diphosphate synthase
("gpps"). A DNA fragment comprising a nucleotide sequence encoding
the geranyl diphosphate synthase was generated synthetically using
the coding sequence of the gpps gene of Arabidopsis thaliana
(GenBank accession number Y17376, REGION: 52 . . . 1320),
codon-optimized for expression in Escherichia coli, as a template.
The nucleotide sequence was flanked by a leader SacII restriction
enzyme site and a terminal SacI restriction enzyme site, and can be
cloned into compatible restriction enzyme sites of a cloning vector
such as a standard pUC or pACYC origin vector. The synthetically
generated geranyl diphosphate synthase sequence was isolated by
digesting the DNA synthesis construct to completion using SacII and
SacI restriction enzymes, resolving the reaction mixture by gel
electrophoresis, gel extracting the approximately 1.3 kb DNA
fragment, and ligating the isolated DNA fragment into the SacII
SacI restriction enzyme site of expression plasmid pMBIS, yielding
expression plasmid pMBIS-gpps (see FIG. 3 for a plasmid map).
[0243] Expression plasmid pAM45 was generated by inserting the MBIS
operon into pAM36-MevT66 and adding lacUV5 promoters in front of
the two operons. The MBIS operon was PCR amplified from pMBIS using
primers comprising a 5' XhoI restriction enzyme site and a 3' PacI
restriction enzyme site. The amplified PCR product was digested to
completion using XhoI and PacI restriction enzymes, the reaction
mixture was resolved by gel electrophoresis, the 5.4 kb DNA
fragment was gel extracted, and the isolated DNA fragment was
ligated into the XhoI PacI restriction enzyme site of pAM36-MevT66,
yielding plasmid pAM43. A DNA fragment comprising a nucleotide
sequence encoding the lacUV5 promoter was synthesized from
oligonucleotides and sub-cloned into the AscI SfiI and AsiSI XhoI
restriction enzyme sites of pAM43, yielding expression plasmid
pAM45.
Example 2
[0244] This example describes methods for making expression vectors
encoding enzymes including enzymes of the MEV pathway from
Staphylococcus aureus organized in operons.
[0245] Expression plasmid pAM41 was derived from expression plasmid
pAM25 by replacing the coding sequence of the HMG1 gene, which
encodes the Saccharomyces cerevisiae HMG-CoA reductase, with the
coding sequence of the mvaA gene, which encodes the Staphylococcus
aureus HMG-CoA reductase (GenBank accession number BA000017,
REGION: 2688925 . . . 2687648). The coding sequence of the mvaA
gene was PCR amplified from Staphylococcus aureus subsp. aureus
(ATCC 70069) genomic DNA using primers 4-49 mvaA SpeI (SEQ ID NO:
2) and 4-49 mvaAR XbaI (SEQ ID NO: 3), the amplified DNA fragment
was digested to completion using SpeI restriction enzyme, the
reaction mixture was resolved by gel electrophoresis, and the
approximately 1.3 kb DNA fragment was gel extracted. The HMG1
coding sequence was removed from pAM25 by digesting the plasmid to
completion using HindIII restriction enzyme. The terminal overhangs
of the resulting linear DNA fragment were blunted using T4 DNA
polymerase. The DNA fragment was then partially digested using SpeI
restriction enzyme, the reaction mixture was resolved by gel
electrophoresis, and the 4.8 kb DNA fragment was gel extracted. The
isolated DNA fragment was ligated with the SpeI-digested mvaA PCR
product, yielding expression plasmid pAM41. The nucleotide sequence
of the atoB(opt):ERG13(opt):mvaA operon contained in pAM41 is SEQ
ID NO: 41.
[0246] Expression plasmid pAM52 was derived from expression plasmid
pAM41 by replacing the coding sequence of the ERG13 gene, which
encodes the Saccharomyces cerevisiae HMG-CoA synthase, with the
coding sequence of the mvaS gene, which encodes the Staphylococcus
aureus HMG-CoA synthase (GenBank accession number BA000017, REGION:
2689180 . . . 2690346). ERG13 is also known as HMGS or HMG-CoA
synthase. The coding sequence of the mvaS gene was PCR amplified
from Staphylococcus aureus subsp. aureus (ATCC 70069) genomic DNA
using primers HMGS 5' Sa mvaS-S (SEQ ID NO: 4) and HMGS 3' Sa
mvaS-AS (SEQ ID NO: 5), and the amplified DNA fragment was used as
a PCR primer to replace the coding sequence of the HMG1 gene in
pAM41 according to the method of Geiser et al. (BioTechniques
31:88-92 (2001)), yielding expression plasmid pAM52. The nucleotide
sequence of the atoB(opt):mvaS:mvaA operon contained in pAM52 is
SEQ ID NO: 42.
[0247] Expression plasmid pAM97 was derived from expression plasmid
pAM45 by replacing the MevT66 operon with the (atoB(opt):mvaS:mvaA)
operon of expression plasmid pAM52. Expression plasmid pAM45 was
digested to completion using AsiSI and SfiI restriction enzymes,
the reaction mixture was resolved by gel electrophoresis, and the
8.3 kb DNA fragment lacking the MevT66 operon was gel extracted.
The (atoB(opt):mvaS:mvaA) operon of pAM52 was PCR amplified using
primers 19-25 atoB SfiI-S (SEQ ID NO: 6) and 19-25 mvaA-AsiSI-AS
(SEQ ID NO: 7), the PCR product was digested to completion using
SfiI and AsiSI restriction enzymes, the reaction mixture was
resolved by gel electrophoresis, the 3.7 kb DNA fragment was gel
extracted, and the isolated DNA fragment was ligated into the AsiSI
SfiI restriction enzyme site of expression plasmid pAM45, yielding
expression plasmid pAM97.
[0248] Expression plasmid pAM97-MBI was derived from expression
plasmid pAM97 and pAM45 by replacing the MBIS operon of pAM97 with
the MBI operon of pAM45. The MBI operon was PCR amplified from
pAM45 using primers 9-70C (SEQ ID NO: 8) and 26-39B (SEQ ID NO: 9),
the reaction mixture was resolved by gel electrophoresis, the 4.5
kb DNA fragment was gel extracted, and the isolated DNA fragment
was digested to completion using SacI and XhoI restriction enzymes.
Expression plasmid pAM97 was digested to completion using SacI and
XhoI restriction enzymes, the reaction mixture was resolved by gel
electrophoresis, the 7.6 kb fragment was gel extracted, and the
isolated DNA fragment was ligated with the MBI operon PCR product,
yielding expression plasmid pAM97-MBI.
[0249] Expression plasmid pAM97-MevB was derived from expression
plasmid pAM97 and pAM45 by replacing the MBIS operon of pAM97 with
the MevB operon of pAM45. The MevB operon was PCR amplified from
pAM45 using primers 9-70C (SEQ ID NO: 8) and 26-39A (SEQ ID NO:
10), the reaction mixture was resolved by gel electrophoresis, the
3.9 kb DNA fragment was gel extracted, and the isolated DNA
fragment was digested to completion using SacI and XhoI restriction
enzymes. Expression plasmid pAM97 was digested to completion using
SacI and XhoI restriction enzymes, the reaction mixture was
resolved by gel electrophoresis, the 7.6 kb fragment was gel
extracted, and the isolated DNA fragment was ligated with the MevB
operon PCR product, yielding expression plasmid pAM97-MevB.
[0250] Expression plasmid pAM128 was generated by inserting the
(atoB(opt):mvaS:mvaA) and MBIS operons of expression plasmid pAM97
into a vector that comprises the RK2 plasmid replication,
segregation, and maintenance system, which obviates the continuous
need for antibiotic selection of host cell transformants. The RK2
plasmid was digested to completion using PstI restriction enzyme,
the reaction mixture was resolved by gel electrophoresis, the
approximately 6.3 kb DNA fragment containing the entire par locus
was gel extracted, and the isolated DNA fragment was subcloned into
the PstI restriction enzyme site of the mini RK2 replicon pRR10
(Roberts et al. (1990) J Bacteriol. 172(11): 6204-6216), yielding
vector pAM132. Expression plasmid pAM97 was digested to completion
using AscI and SacI restriction enzymes, the reaction mixture was
resolved by gel electrophoresis, the approximately 9.4 kb DNA
fragment was gel extracted, and the isolated DNA fragment was
ligated into the MluI SacI restriction enzyme site of pAM132,
yielding expression plasmid pAM128.
Example 3
[0251] This example describes methods for making expression vectors
that encode enzymes including enzymes of the MEV pathway from
Enterococcus faecalis organized in operons.
[0252] Plasmid pAM16 was generated by inserting the coding sequence
of the mvaE gene of Enterococcus faecalis (GenBank accession number
AF290092 REGION: 1479 . . . 3890) (encodes an acetyl-CoA
acetyltransferase/HMG-CoA reductase (HMGR)) into the
pBlueScripII-KS(+) vector. The coding sequence of the mvaE gene was
PCR amplified from Enterococcus faecalis genomic DNA (ATCC 700802)
using 5' phosphorylated primers 4-40 mvaEF BamHI (SEQ ID NO: 11)
and 4-40 mvaERHindIII (SEQ ID NO: 12). (Note that primer 4-40 mvaEF
BamHI changes the start codon of the mvaE gene from TTG to ATG in
the amplified PCR product.) The resulting PCR product was ligated
into the SmaI restriction enzyme site of pBlueScripII-KS(+)
(Stratagene, La Jolla, Calif.), yielding expression plasmid
pAM16.
[0253] Plasmid pAM18 was generated by inserting the coding sequence
of the mvaS gene of Enterococcus faecalis (GenBank accession number
AF290092 REGION: 142 . . . 1293) (encodes a HMG-CoA synthase
(HMGS)) into the pBlueScripII-KS(+) vector. The coding sequence of
the mvaS gene was PCR amplified from Enterococcus faecalis genomic
DNA (ATCC 700802) using 5' phosphorylated primers 4-40 mvaSF BgIII
(SEQ ID NO: 13) and 4-39 mvaSR BamHI (SEQ ID NO: 14), and the PCR
product was ligated into the SmaI restriction enzyme site of
pBlueScripII-KS(+) (Stratagene, La Jolla, Calif.), yielding
expression plasmid pAM18.
[0254] Expression plasmid pAM22 was generated by inserting the
coding sequence of the mvaE gene of expression plasmid pAM16 into
the pZE21-P.sub.L-laCO1 vector. Vector pZE.sup.21-P.sub.L-lacO1 is
a derivative of vector pZE21-MCS-1 in which the tet promoter was
replaced with the P.sub.L-lacO1 promoter (Lutz and Bujard (1997)
Nucl Acids Res. 25:1203-1210). Expression plasmid pAM16 was
digested to completion using BamHI and HindIII restriction enzymes,
the reaction mixture was resolved by gel electrophoresis, the
approximately 2.4 kb DNA fragment containing the mvaE coding
sequence was gel extracted, and the isolated DNA fragment was
inserted into the BamHI HindIII restriction enzyme site of
pZE21-P.sub.L-lacO1, yielding expression plasmid pAM22.
[0255] Expression plasmid pAM33 was generated by inserting the
coding sequence of the mvaS gene of expression plasmid pAM18 into
expression plasmid pAM22. Expression plasmid pAM18 was digested to
completion using BglII and BamHI restriction enzymes, the reaction
mixture was resolved by gel electrophoresis, the approximately 1.2
kb DNA fragment containing the coding sequence of the mvaS gene was
gel extracted, and the isolated DNA fragment was inserted into the
BamHI site of expression plasmid pAM22, yielding expression plasmid
pAM33.
[0256] Expression plasmid pAM34 was generated by inserting the
mvaS-mvaE operon of expression plasmid pAM33 into vector pAM29. The
mvaS-mvaE operon was isolated by partially digesting pAM33 using
EcoRI restriction enzyme, digesting the resulting linear DNA
fragment using MluI restriction enzyme, resolving the reaction
mixture by gel electrophoresis, and gel extracting the
approximately 3.6 kb DNA fragment. The vector backbone of pAM29 was
obtained by digesting to completion expression vector pAM25 using
MluI and EcoRI restriction enzymes, resolving the reaction mixture
by gel electrophoresis, and gel extracting the approximately 2.1 kb
DNA fragment. The two isolated DNA fragments were ligated, yielding
expression plasmid pAM34.
Example 4
[0257] This example describes methods for making expression
plasmids that encode enzymes, including enzymes of the DXP pathway
from Escherichia coli organized in operons.
[0258] Expression plasmid pAM408 was generated by inserting genes
encoding enzymes of the "top" DXP pathway into the pAM29 vector.
Enzymes of the "top" DXP pathway include
1-deoxy-D-xylulose-5-phosphate synthase (encoded by the dxs gene of
Escherichia coli), 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(encoded by the dxr gene of Escherichia coli),
4-diphosphocytidyl-2C-methyl-D-erythritol synthase (encoded by the
ispD gene of Escherichia coli), and
4-diphosphocytidyl-2C-methyl-D-erythritol synthase (encoded by the
ispE gene of Escherichia coli), which together transform pyruvate
and D-glyceraldehyde-3-phosphate to
4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. DNA
fragments comprising nucleotide sequences that encode enzymes of
the "top" DXP pathway were generated by PCR amplifying the coding
sequences of the dxs (GenBank accession number U00096 REGION:
437539 . . . 439401), dxr (GenBank accession number U00096 REGION:
193521 . . . 194717), ispD (GenBank accession number U00096 REGION:
2869803 . . . 2870512), and ispE (GenBank accession number U00096
REGION 1261249 . . . 12621100) genes from Escherichia coli strain
DH1 (ATCC #33849) with added optimal Shine Dalgarno sequences and
5' and 3' restriction enzyme sites using the PCR primers shown in
SEQ ID NOS: 15-18. The PCR products were resolved by gel
electrophoresis, gel extracted using a Qiagen (Valencia, Calif.)
gel purification kit, digested to completion using appropriate
restriction enzymes (XhoI and KpnI for the PCR product comprising
the dxs gene; KpnI and ApaI for the PCR product comprising the dxr
gene; ApaI and NdeI for the PCR product comprising the ispD gene;
NdeI and MluI for the PCR product comprising the ispE gene), and
purified using a Qiagen (Valencia, Calif.) PCR purification kit.
Roughly equimolar amounts of each PCR product were then added to a
ligation reaction to assemble the individual genes into an operon.
From this ligation reaction, 1 .mu.l of reaction mixture was used
to PCR amplify 2 separate gene cassettes, namely the dxs-dxr and
the ispD-ispE gene cassettes. The dxs-dxr gene cassette was PCR
amplified using primers 67-1A-C (SEQ ID NO: 15) and 67-1D-C (SEQ ID
NO: 18), and the ispD-ispE gene cassette was PCR amplified using
primers 67-1E-C (SEQ ID NO: 19) and 67-1H-C (SEQ ID NO: 22). The
two PCR products were resolved by gel electrophoresis, and gel
extracted. The PCR product comprising the dxs-dxr gene cassette was
digested to completion using XhoI and ApaI restriction enzymes, and
the PCR product comprising the ispD-ispE gene cassette was digested
to completion using ApaI and MluI restriction enzymes, and the two
PCR products were purified. Vector pAM29 was digested to completion
using SalI and MluI restriction enzymes, and the two digested PCR
products containing the "top" DXP pathway operon were ligated into
the SalI MluI restriction enzyme site of the pAM29 vector, yielding
expression plasmid pAM408 (see FIG. 4 for a plasmid map).
[0259] Expression plasmid pAM409 was generated by inserting genes
encoding enzymes of the "bottom" DXP pathway into the pAM369
vector. Enzymes of the "bottom" DXP pathway include
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (encoded by
the ispF gene of Escherichia coli),
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (encoded by
the ispG gene of Escherichia coli), and isopentenyl/dimethylallyl
diphosphate synthase (encoded by the ispH gene of Escherichia
coli), which together transform
4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to IPP and
DMAPP. IPP is also converted to DMAPP through the activity of
isopentyl diphosphate isomerase (encoded by the idi gene of
Escherichia coli). DMAPP can be further converted to FPP through
the activity of farnesyl diphosphate synthase (encoded by the ispA
gene of Escherichia coli). An operon encoding enzymes of the
"bottom" DXP pathway as well as an isopentyl diphosphate isomerase
and a farnesyl diphosphate synthase was generated by PCR amplifying
the ispF (GenBank accession number U00096 REGION: 2869323 . . .
2869802), ispG (GenBank accession number U00096 REGION: 2638708 . .
. 2639826), ispH (GenBank accession number U00096 REGION: 26277 . .
. 27227), idi (GenBank accession number AF119715), and ispA
(GenBank accession number D00694 REGION: 484 . . . 1383) genes from
Escherichia coli strain DH1 (ATCC #33849) with added optimal Shine
Dalgarno sequences and 5' and 3' restriction enzyme sites using the
appropriate PCR primers. The PCR products were resolved by gel
electrophoresis, gel extracted, digested with the appropriate
restriction enzymes (BamHI and ApaI for the PCR product comprising
the ispF gene; KpnI and ApaI for the PCR product comprising the
ispG gene; SalI and KpnI for the PCR product comprising the ispH
gene; SalI and HindIII for the PCR product comprising the idi gene;
HindIII and NcoI for the PCR product comprising the ispA gene), and
purified. Roughly equimolar amounts of each PCR product were then
added to a ligation reaction to assemble the individual genes into
an operon. From this ligation reaction, 1 .mu.l of reaction mixture
was used to PCR amplify 2 separate gene cassettes, namely the
ispF-ispG and the ispH-idi-ispA gene cassettes. The ispF-ispG gene
cassette was PCR amplified using primers 67-2A-C (SEQ ID NO: 23)
and 67-2D-C (SEQ ID NO: 26), and the ispH-idi-ispA gene cassette
was PCR amplified using primers 67-2E-C (SEQ ID NO: 27) and 67-2J-C
(SEQ ID NO: 32). The two PCR products were resolved by gel
electrophoresis, and gel extracted. The PCR product comprising the
ispF-ispG gene cassette was digested to completion using BamHI and
KpnI restriction enzymes, and the PCR product comprising the
ispH-idi-ispA gene cassette was digested to completion using KpnI
and NcoI restriction enzymes, and the two PCR products were
purified. Vector pAM369 was created by assembling the p15A origin
of replication from pAM29 and beta-lactamase gene for ampicillin
resistance from pZE12-luc (Lutz and Bujard (1997) Nucl Acids Res.
25:1203-1210) with an oligonucleotide-generated lacUV5 promoter.
Vector pAM369 was digested to completion using BamHI and NcoI
restriction enzymes, and the 2 isolated PCR products containing the
"bottom" DXP pathway operon were ligated into the BamHI NcoI
restriction enzyme site of the pAM369 vector, yielding expression
plasmid pAM409.
[0260] Expression plasmid pAM424, a derivative of expression
plasmid pAM409 containing the broad-host range RK2 origin of
replication, was generated by transferring the lacUV5 promoter and
the ispFGH-idi-ispA operon of pAM409 to the pAM257 vector. Vector
pAM257 was generated as follows: the RK2 par locus was
PCR-amplified from RK2 plasmid DNA (Meyer et al. (1975) Science
190:1226-1228) using primers 9-156A (SEQ ID NO: 33) and 9-156B (SEQ
ID NO: 34), the 2.6 kb PCR product was digested to completion using
AatII and XhoI restriction enzymes, and the DNA fragment was
ligated into a plasmid containing the p15 origin of replication and
the chloramphenicol resistance gene from vector pZA31-luc (Lutz and
Bujard (1997) Nucl Acids Res. 25:1203-1210), yielding plasmid
pAM37-par; pAM37-par was digested to completion using restriction
enzymes SacI and HindIII, the reaction mixture was resolved by gel
electrophoresis, the DNA fragment comprising the RK2 par locus and
the chloramphenicol resistance gene was gel extracted, and the
isolated DNA fragment was ligated into the SacI HindIII site of the
mini-RK2 replicon pRR10 (Roberts et al. (1990) J Bacteriol.
172:6204-6216), yielding vector pAM133; pAM133 was digested to
completion using BglII and HindIII restriction enzymes, the
reaction mixture was resolved by gel electrophoresis, the
approximately 6.4 kb DNA fragment lacking the ampicillin resistance
gene and oriT conjugative origin was gel extracted, and the
isolated DNA fragment was ligated with a synthetically generated
DNA fragment comprising a multiple cloning site that contained PciI
and XhoI restriction enzyme sites, yielding vector pAM257.
Expression plasmid pAM409 was digested to completion using XhoI and
PciI restriction enzymes, the reaction mixture was resolved by gel
electrophoresis, and the approximately 4.4 kb DNA fragment was gel
extracted. Vector pAM257 was digested to completion using
restriction enzymes XhoI and PciI, and the isolated DNA fragment
containing the lacUV5 promoter and ispFGH-idi-ispA operon was
ligated into the XhoI PciI restriction enzyme site of the pAM257
vector, yielding expression plasmid pAM424 (see FIG. 5 for a
plasmid map).
Example 5
[0261] This example describes methods for making expression
plasmids that encode enzymes that convert FPP or GPP.
[0262] Expression plasmid pTrc99A-ADS was generated by inserting a
nucleotide sequence encoding an amorpha-4,11-diene synthase ("ADS")
into vector pTrc99A. The amorpha-4,11-diene synthase sequence was
generated synthetically, so that upon translation the amino acid
sequence would be identical to that described by Merke et al.
(2000) Ach. Biochem. Biophys. 381:173-180, so that the nucleotide
sequence encoding the amorpha-4,11-diene synthase was optimized for
expression in Escherichia coli, and so that the nucleotide sequence
was flanked by a 5' NcoI and a 3' XmaI restriction enzyme site (see
U.S. Pat. No. 7,192,751). The nucleotide sequence was digested to
completion using NcoI and XmaI restriction enzymes, the reaction
mixture was resolved by gel electrophoresis, the approximately 1.6
kb DNA fragment was gel-extracted, and the isolated DNA fragment
was inserted into the NcoI XmaI restriction enzyme site of the
pTrc99A vector (Amman et al. (1985) Gene 40:183-190), yielding
expression plasmid pTrc99A-ADS (see FIG. 6 for a plasmid map).
[0263] Expression plasmid pAM113 is a chloramphenicol-resistant
derivative of pTrc99A-ADS. It was generated by PCR amplifying the
chloramphenicol resistance gene from vector pZA31-luc (Lutz and
Bujard (1997) Nucl Acids Res. 25:1203-1210) using 5'-phosphorylated
primers 19-137 cml-pAM37-AS (SEQ ID NO: 35) and 19-137 cml-pAM37-S
(SEQ ID NO: 36), and inserting the 920 bp PCR product into the FspI
restriction enzyme site of expression plasmid pTrc99A-ADS, yielding
expression plasmid pAM113.
[0264] Expression plasmid pC9 was generated by inserting a genomic
DNA fragment of Bacillus subtilis 6051 comprising the coding
sequence of the nudF gene and upstream genomic sequences (GenBank
accession number Z99116 REGION: 49364 . . . 48548) into vector
pTrc99A (Amann et al. (1988) Gene 69:301-315). Expression plasmid
pNudF-H was generated by inserting the coding sequence of the
Bacillus subtilis 6051 nudF gene (GenBank accession number Z99116
REGION: 49105 . . . 48548) into vector pTrc99A. Expression plasmid
pyhfR was generated by inserting the coding sequence of the
Bacillus subtilis 6051 yhjR gene (GenBank accession number Z99109
REGION: 97583 . . . 97002) into vector pTrc99A.
[0265] Expression plasmid pAM373 was generated by inserting a
nucleotide sequence encoding the .beta.-farnesene synthase ("FSB")
of Artemisia annua (GenBank accession number AY835398),
codon-optimized for expression in Escherichia coli, into the
pTrc99A vector. The nucleotide sequence encoding the
.beta.-farnesene synthase was generated synthetically, and was
amplified by PCR from its DNA synthesis construct using the
appropriate primers. To create a leader NcoI restriction enzyme
site in the PCR product comprising the .beta.-farnesene synthase
coding sequence, the codon encoding the second amino acid in the
original polypeptide sequence (TCG coding for serine) was replaced
by a codon encoding aspartic acid (GAC) in the 5' PCR primer (SEQ
ID NO: 37). The resulting PCR product was partially digested using
NcoI restriction enzyme, and digested to completion using SacI
restriction enzyme, the reaction mixture was resolved by gel
electrophoresis, the approximately 1.7 kb DNA fragment comprising
the .beta.-farnesene synthase coding sequence was gel extracted,
and the isolated DNA fragment was ligated into the NcoI SacI
restriction enzyme site of the pTrc99A vector, yielding expression
plasmid pAM373 (see FIG. 6 for a plasmid map).
[0266] Expression plasmids pTrc99A-FSA, pTrc99A-GTS, pTrc99A-PS,
pTrc99A-TS were generated by inserting a DNA fragment comprising a
nucleotide sequence encoding an .alpha.-farnesene synthase ("FSA"),
a .gamma.-terpinene synthase ("GTS"), an .alpha.-pinene synthase
("APS"), or a terpinolene synthase ("TS") into the pTrc99A vector.
The DNA fragment insert was generated synthetically, using as a
template for example the coding sequence of the .alpha.-farnesene
synthase gene of Picea abies (GenBank accession number AY473627,
REGION: 24 . . . 1766), the coding sequence of the .beta.-farnesene
synthase gene of Artemisia annua (GenBank accession number
AY835398), the coding sequence of the .gamma.-terpinene synthase
gene of Citrus limon (GenBank accession number AF514286 REGION: 30
. . . 1832), the coding sequence of the .alpha.-pinene synthase
gene of Abies grandis (GenBank accession number U87909, REGION: 6 .
. . 1892) or of Pinus taeda (GenBank accession number AF543530
REGION: 1 . . . 1887), or the coding sequence of the terpinolene
synthase gene of Ocimum basilicum (GenBank accession number
AY693650) or of Pseudotsuga menziesii (GenBank accession number
AY906866 REGION: 10 . . . 1887) or of Abies grandis (GenBank
accession number AF139206), all nucleotide sequences being
codon-optimized for expression in Escherichia coli. The DNA
fragments for FSA was amplified by PCR from its DNA synthesis
construct using the primer sequences SEQ ID NO: 39 and SEQ ID NO:
40. The resulting PCR product was digested to completion using NcoI
and SacI restriction enzymes, the reaction mixture was resolved by
gel electrophoresis, the approximately 1.7 kb DNA fragment
comprising the .alpha.-farnesene synthase coding sequence was gel
extracted, and the isolated DNA fragment was ligated into the NcoI
SacI restriction enzyme site of the pTrc99A vector, yielding
expression plasmid pTrc99A-FSA (see FIG. 6 for a plasmid map). The
DNA fragments for GTS, APS, and TS were designed to be flanked by a
leader XmaI restriction enzyme site and a terminal XbaI restriction
enzyme site, and were cloned into compatible restriction enzyme
sites of a cloning vector such as a standard pUC or pACYC origin
vector, from which they could be liberated again by digesting to
completion the DNA synthesis construct using XbaI and XmaI
restriction enzymes, resolving the reaction mixture by gel
electrophoresis, and gel extracting the 1.7 to 1.9 terpene synthase
encoding DNA fragment. The isolated DNA fragments were ligated into
the XmaI XbaI restriction enzyme site of vector pTrc99A (Amman et
al., Gene 40:183-190 (1985)), yielding plasmids pTrc99A-GTS,
pTrc99A-APS, or pTrc99A-TS (see FIG. 6 for plasmid maps).
[0267] Expression plasmids pRS425-FSA and pRS425-FSB were generated
by inserting a nucleotide sequence encoding an .alpha.-farnesene
synthase ("FSA") or a .beta.-farnesene synthase ("FSB"),
respectively, into the pRS425-Gal1 vector (Mumberg et. al. (1994)
Nucl. Acids. Res. 22(25): 5767-5768). The nucleotide sequence
inserts were generated synthetically, using as a template for
example the coding sequence of the .alpha.-farnesene synthase gene
of Picea abies (GenBank accession number AY473627, REGION: 24 . . .
1766) or of the .beta.-farnesene synthase gene of Artemisia annua
(GenBank accession number AY835398), codon-optimized for expression
in Saccharomyces cerevisiae. The synthetically generated nucleotide
sequence was flanked by a 5' BamHI site and a 3' XhoI site, and
could thus be cloned into compatible restriction enzyme sites of a
cloning vector such as a standard pUC or pACYC origin vector. The
synthetically generated nucleotide sequence was isolated by
digesting to completion the DNA synthesis construct using BamHI and
XhoI restriction enzymes. The reaction mixture was resolved by gel
electrophoresis, the approximately 1.7 kb DNA fragment comprising
the .alpha.-farnesene synthase or .beta.-farnesene synthase coding
sequence was gel extracted, and the isolated DNA fragment was
ligated into the BamHI XhoI restriction enzyme site of the
pRS425-Gal1 vector, yielding expression plasmid pRS425-FSA or
pRS425-FSB, respectively.
[0268] Expression plasmids pTrc99A-LLS, pTrc99A-LMS, pTrc99A-BPS,
pTrc99A-PHS, pTrc99A-CS, and pTrc99A-SS are generated by inserting
a nucleotide sequence encoding a linalool synthase ("LLS"),
limonene synthase ("LMS"), .beta.-pinene synthase ("BPS"),
.beta.-phellandrene ("PHS"), carene synthase ("CS"), or sabinine
synthase ("SS") into the pTrc99A vector. The nucleotide sequence
inserts are generated synthetically, using as a template for
example the coding sequence of the linalool synthase gene of
Artemisia annua (GenBank accession number AF154124, REGION: 13 . .
. 1764), the coding sequence of the limonene synthase gene of Abies
grandis (GenBank accession number AF006193 REGION: 73 . . . 1986),
the coding sequence of the .beta.-pinene synthase of Artemisia
annua (GenBank accession number AF276072 REGION: 1 . . . 1749), the
coding sequence of the .beta.-phellandrene synthase gene of Abies
grandis (GenBank accession number AF139205 REGION: 34 . . . 1926),
the coding sequence of the carene synthase gene of Salvia
stenophylla (GenBank accession number AF527416 REGION: 78 . . .
1871), or the coding sequence of the sabinene synthase gene of
Salvia officinalis (GenBank accession number AF051901 REGION: 26 .
. . 1798). The nucleotide sequences encoding the .beta.-pinene,
sabinine, and .beta.-phellandrene synthases are flanked by a leader
XmaI restriction enzyme site and a terminal XbaI restriction enzyme
site, the nucleotide sequences encoding the linalool and carene
synthases are flanked by a leader NcoI restriction enzyme site and
a terminal XmaI restriction enzyme site, and the nucleotide
sequence encoding the limonene synthase is flanked by a leader NcoI
restriction enzyme site and a terminal PstI restriction enzyme
site. The DNA synthesis constructs are digested to completing using
XmaI and XbaI (for the .beta.-pinene, sabinine, and
.beta.-phellandrene synthase constructs), NcoI and XmaI restriction
enzymes (for the linalool and careen synthase constructs), or XbaI
and PstI restriction enzymes (for the limonene synthase construct).
The reaction mixtures are resolved by gel electrophoresis, the
approximately 1.7 to 1.9 kb DNA fragments are gel extracted, and
the isolated DNA fragments are ligated into the XmaI XbaI
restriction enzyme site (for the .beta.-pinene, sabinine, and
.beta.-phellandrene synthase inserts), the NcoI XmaI restriction
enzyme site (for the linalool and carene synthase inserts), or the
XbaI PstI restriction enzyme site (for the limonene synthase
insert) of the pTrc99A vector, yielding expression plasmids
pTrc99A-LLS, pTrc99A-LMS, pTrc99A-BPS, pTrc99A-PHS, pTrc99A-CS, and
pTrc99A-SS (see FIG. 6 for plasmid maps).
Example 6
[0269] This example describes the generation of Escherichia coli
host strains useful in the invention.
[0270] As detailed in Table 1, the host strains were created by
transforming chemically competent Escherichia coli parent cells
with one or more expression plasmids of Example 1 through 5.
TABLE-US-00001 TABLE 1 E. coli host strains Host Strain E. coli
Parent Strain Expression Plasmids Antibiotic Selection B32 DH1
pMevT 100 ug/mL carbenicillin B292 B pMBIS 5 ug/mL tetracycline
B210 DP pTrc99A-ADS 34 ug/mL chloramphenicol B153 DH1 pAM97 100
ug/mL carbenicillin B282 DP pTrc99A-ADS 34 ug/mL chloramphenicol
B255 DH1 pAM128 100 ug/mL carbenicillin B256 DP pAM113 34 ug/mL
chloramphenicol B86 DH1 pAM52 50 ug/mL kanamycin pMBIS 100 ug/mL
carbenicillin pTrc99A-ADS 5 ug/mL tetracycline B61 DH1 pAM25
pBBR1MCS-3 pTrc99A B62 pAM34 pBBR1MCS-3 pTrc99A B003 DH10B
pTrc99A-ADS 100 .mu.g/ml carbenicillin B617 pAM408 100 ug/mL
carbenicillin pTrc99A-ADS 50 ug/mL kanamycin B618 pAM424 100 ug/mL
carbenicillin pTrc99A-ADS 35 .mu.g/ml chloramphenicol B619 pAM408
100 .mu.g/ml carbenicillin pAM424 50 .mu.g/ml kanamycin pTrc99A-ADS
35 .mu.g/ml chloramphenicol B650 DH10B pAM373 100 .mu.g/ml
carbenicillin B651 pAM408 100 .mu.g/ml carbenicillin pAM373 50
.mu.g/ml kanamycin B652 pAM424 100 .mu.g/ml carbenicillin pAM373 35
.mu.g/ml chloramphenicol B653 pAM408 100 .mu.g/ml carbenicillin
pAM424 50 .mu.g/ml kanamycin pAM373 35 .mu.g/ml chloramphenicol
B286 DH1 pAM97-MevB 100 ug/mL carbenicillin pC9 34 ug/mL B287
pAM97-MevB chloramphenicol. pnudF-H B288 pAM97-MevB pyhfR B291
pAM97-MBI pyhfR B592 DH1 pMevT 100 ug/mL carbenicillin pMBIS 34
ug/mL pTrc99A-FSA chloramphenicol B552 pMevT 5 ug/mL tetracycline
pMBIS pAM373 Example 21 host cell pMevT (production of GTS, APS,
pMBIS-gpps TS) pTrc99A-GTS or -APS or -TS Example 21 host cell
pMevT 100 ug/mL carbenicillin (production of LLS, LMS, pMBIS-gpps
34 ug/mL BPS, PHS, CS, SS) pTrc99A-LLS or chloramphenicol -LMS or
-BPS or 5 ug/mL tetracycline -PHS or -CS or -SS
[0271] Host cell transformants were selected on Luria Bertoni (LB)
agar containing antibiotics as detailed in Table 1. Single colonies
were transferred from LB agar to culture tubes containing 5 mL of
LB liquid medium and antibiotics. B003, B617, B618, B619, B650,
B651, B652, and B653 host cell transformants were incubated at
30.degree. C. on a rotary shaker at 250 rpm for 30 hours. All other
host cell transformants were incubated at 37.degree. C. on a rotary
shaker at 250 rpm until growth reached stationary phase. The cells
were adapted to minimal media by passaging them through 4 to 5
successive rounds of M9-MOPS media containing 0.8% glucose and
antibiotics (see Table 2 for the composition of the M9-MOPS
medium). The cells were stored at -80.degree. C. in cryo-vials in 1
mL stock aliquots made up of 400 uL sterile 50% glycerol and 600 uL
liquid culture.
TABLE-US-00002 TABLE 2 Composition of M9-MOPS Culture Medium
Component Quantity (per L) Na2HPO47H2O 12.8 g KH2PO4 3 g NaCl 0.5 g
NH4Cl 1 g MgSO4 2 mmol CaCl2 0.1 mmol Thiamine 0.1 ug MOPS buffer
pH 7.4 100 mmol (NH3)6Mo7O244H2O 3.7 ug H3BO4 25 ug CoCl2 7.1 ug
CuSO4 2.4 ug MnCl2 16 ug ZnSO4 2.9 ug FeSO4 0.28 mg
Example 7
[0272] This example demonstrates expression plasmid stability in
the absence of antibiotics in an Escherichia coli host strain that
harbors an expression plasmid comprising the RK2 plasmid
replication, segregation, and maintenance system.
[0273] A seed culture of host strain B255 was established by adding
a stock aliquot of the strain to a 125 mL flask containing 40 mL
M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics as
detailed in Table 1, and by growing the culture overnight.
[0274] The seed culture was used to inoculate at an initial
OD.sub.600 of approximately 0.05, two 250 mL flasks each containing
40 mL M9-MOPS medium, 2% glucose, and 0.5% yeast extract. Culture
#1 also contained 100 ug/mL carbenicillin and 34 ug/1 mL
chloramphenicol. Culture #2 did not receive any antibiotics. Both
cultures were incubated at 37.degree. C. on a rotary shaker at 250
rpm until they reached an OD.sub.600 of approximately 0.2, at which
point the production of amorpha-4,11-diene in the host cells was
induced by adding 40 uL of 1M IPTG to the culture medium. At the
time of induction, the cultures were overlain with 8 mL of an
organic overlay to capture the amorpha-4,11-diene. Samples were
taken periodically for a total of 72 hours. Production of
amorpha-4,11-diene by the host strain in the 2 cultures was
confirmed by GC/MS as described in Example 10.
[0275] To assess plasmid stability in the two cell cultures, a
sample of each culture was removed at 72 hours and streaked onto a
LB agar plate (no antibiotics). After overnight incubation at
37.degree. C., 50 individual colonies derived from each culture
were replica-plated onto a LB agar-plus-antibiotics (34 ug/mL
chloramphenicol, 100 ug/mL carbenicillin) plate and a LB
agar-minus-antibiotics (no antibiotic) plate. After another
overnight incubation at 37.degree. C., the LB agar-plus-antibiotics
and the LB agar-minus-antibiotics plate were each found to contain
approximately 50 colonies, indicating that plasmid retention both
in the presence and in the absence of antibiotics in the culture
medium had been approximately 100%.
Example 8
[0276] This example demonstrates increased specific activity and
stability of the Enterococcus faecalis HMGR compared to the
Saccharomyces cerevisiae tHMGR in an Escherichia coli host
strain.
[0277] Seed cultures of host strains B61 and B62 were established
by adding a stock aliquot of each strain to 125 mL flasks
containing 20 mL M9-MOPS medium, 0.8% % glucose, and antibiotics as
detailed in Table 5, and by growing the cultures to saturation. The
seed cultures were diluted 1:100 into 140 mL of fresh medium in a
500 mL flask, and grown again to an OD.sub.550 of approximately
0.1, at which point production of amorpha-4,11-diene was induced by
adding 140 uL 1 M IPTG to each culture. At 4, 12, 20, 28, 36, and
49 hours post-induction, samples were removed from each culture,
and cells were pelleted by centrifugation. The cell pellets were
snap frozen on dry ice, and then stored at -80.degree. C.
[0278] To conduct enzyme assays, cell pellets were thawed on ice,
and then lysed using Bugbuster (Novagen, Madison, Wis.) containing
protease inhibitor mix #3 (Calbiochem, San Diego, Calif.),
benzonase (20 .mu.L oer5 mL bugbuster; Novagen, Madison, Wis.), and
lysozyme (30 ug/mL). Enzyme activity of the Saccharomyces
cerevisiae tHMGR was assayed in 50 mM Tris HCl (pH7.5), 0.2 mM
NADPH (Sigma, St. Louis, Mo.), and 0.3 mM
DL-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) sodium salt
(Sigma, St. Louis, Mo.). The assay was started by adding cell
lysate, and the disappearance of NADPH was monitored by absorbance
at 340 nM. To account for non-specific disappearance of NADPH,
results obtained in a control assay lacking HMG-CoA were subtracted
from results obtained in test samples. Enzyme activity of the
Enterococcus faecalis HMGR was measured similarly except that the
assay buffer contained 100 mM potassium phosphate buffer (pH6.5),
0.4 mM NADPH, 1.0 mM EDTA, and 100 mM KCl.
[0279] Protein assays were done by the method of Bradford ((1976)
Anal Biochem. 72:248-254). Specific activities were calculated as
.DELTA.nmol NADPH/min/mg protein.
[0280] As shown in FIG. 8, the Enterococcus faecalis HMGR exhibited
higher specific activity and increased stability compared to the
Saccharomyces cerevisiae tHMGR.
Example 9
[0281] This example describes the calibration of OD.sub.600 with
dry cell weight ("DCW").
[0282] To obtain the relationship between DCW and OD600, a
representative strain, B32, was grown in high cell density
processes similar to those described in Examples 10-14. Samples
were taken throughout the runs, and the OD.sub.600 and DCW were
measured for each sample. To determine the DCW, the cells were
pelleted and the supernatant discarded. The cell pellet was washed
once with water, and was then dried in an oven at 80.degree. C. for
at least 3 days. The tubes containing cell pellets were weighed,
the weight of the tube was subtracted from the measured weights,
and the remaining weight was divided by the initial volume of each
sample (0.0015 L) to obtain the DCW.
[0283] FIG. 9 shows the relationship between DCW and OD.sub.600
measured in these experiments.
Example 10
[0284] This example demonstrates increased production of
amorpha-4,11-diene in Escherichia coli host strains expressing the
Staphylococcus aureus HMGR and HMGS compared to host strains
expressing the Saccharomyces cerevisiae tHMGR and HMGS.
[0285] Seed cultures of host strains B32, B153, B210, B282, B292,
B86, B255, and B256 were established by adding a stock aliquot of
each strain to separate 125 mL flasks containing 25 mL M9-MOPS
medium, 0.8% glucose, and antibiotics as detailed in Table 1, and
by growing the cultures overnight.
[0286] The seed cultures were used to inoculate at an initial
OD.sub.600 of approximately 0.05 separate 250 mL flasks containing
40 mL M9-MOPS medium, 2% glucose, and antibiotics. The cultures
were incubated at 30.degree. C. on a rotary shaker at 250 rpm until
they reached an OD.sub.600 of approximately 0.2, at which point the
production of amorpha-4,11-diene in the host cells was induced by
adding 40 uL of 1M IPTG to the culture medium. The cultures were
overlain with 8 mL of an organic overlay (e.g., dodecane, methyl
oleate or isopropyl myristate). Samples of the organic overlay
layer and the broth were taken once a day for 72 hours. Broth
samples were used to measure the OD.sub.600. Amorpha-4,11-diene
concentration was measured by transferring 5 uL of the organic
overlay layer to a clean glass vial containing 500 uL ethyl acetate
spiked with beta- or trans-caryophyllene as an internal
standard.
[0287] The organic overlay/ethyl acetate samples were analyzed on a
Hewlett-Packard 6890 gas chromatograph/mass spectrometer (GC/MS) by
scanning only for two ions, the molecular ion (204 m/z) and the 189
m/z ion, as described in Martin et al. (2001) Biotechnol. Bioeng.
75:497-503. To expedite run times, the temperature program and
column matrix was modified to achieve optimal peak resolution and
the shortest overall runtime. A 1 uL sample was separated on the GC
using a DB-XLB column (available from Agilent Technologies, Inc.,
Palo Alto, Calif.) and helium carrier gas. The temperature program
for the analysis was as follows: 100.degree. C. for 0.75 minutes,
increasing temperature at 60.degree. C./minute to a temperature of
300.degree. C., and a hold at 300.degree. C. for 0.5 minutes. The
resolved samples were analyzed by a Hewlett-Packard model 5973
mass-selective detector that monitored ions 189 and 204 m/z.
Previous mass spectra demonstrated that the amorpha-4,11-diene
synthase product was amorpha-4,11-diene, and that
amorpha-4,11-diene had a retention time of 3.7 minutes using this
GC protocol. Beta- or trans-caryophyllene was used as an internal
standard for quantitation. Amorpha-4,11-diene titer was calculated
using the ratio of internal standard to amorpha-4,11-diene peak
areas based upon a quantitative calibration curve of purified
amorpha-4,11-diene (0.63-10 mg/L of KJF17-109-3) in
caryophyllene-spiked ethyl acetate.
[0288] As shown in FIGS. 10A and 10B, strains B153 and B282, which
expressed the Staphylococcus aureus HMGR and HMGS, produced
elevated levels of amorpha-4,11-diene compared to strains B32,
B210, B255, B256, and B292, which expressed the Saccharomyces
cerevisiae tHMGR and HMGS.
Example 11
[0289] This example demonstrates increased production of
amorpha-4,11-diene by an Escherichia coli host strain grown at
suboptimal temperature.
[0290] A seed culture of host strain B32 was established by adding
0.5 mL of a stock aliquot of the strain to a 250 mL flask
containing 50 mL M9-MOPS medium and antibiotics as detailed in
Table 1, and by growing the culture overnight at 37.degree. C. on a
rotary shaker at 250 rpm.
[0291] The seed culture was used to inoculate at an initial
OD.sub.600 of approximately 0.05 four 250 mL flasks, each
containing 40 mL fermentor batch medium (see Table 6 for medium
composition), 100 mM MOPS buffer pH7.1, and antibiotics. The
cultures were incubated on a rotary shaker at 250 rpm at either
30.degree. C. or 37.degree. C. until they reached an OD.sub.600 of
0.18 to 0.22, at which point the production of amorpha-4,11-diene
in the host cells was induced by adding 40 uL of 1M IPTG to the
culture medium. At the time of induction, the cultures were
overlain with 8 mL of an organic overlay to capture the
amorpha-4,11-diene. Samples were taken once a day, and analyzed as
described in Example 10.
[0292] As shown in FIGS. 11A and 11B, fermentation at 30.degree. C.
did not affect cell growth, but led to an approximately 50%
increase in the specific production of amorpha-4,11-diene by the
Escherichia coli host strain.
Example 12
[0293] This example demonstrates increased production of
amorpha-4,11-diene by an Escherichia coli host strain grown under
restricted carbon source conditions.
[0294] A seed culture of host strain B32 for fermentation runs
050608-1 and 050629-1 was established by adding 0.25 uL of a stock
aliquot of the strain to a 250 mL flask containing 50 mL M9-MOPS
medium and antibiotics as detailed in Table 1, and by incubating
the culture at 37.degree. C. on a rotary shaker at 250 rpm until it
reached an OD.sub.600 of 1 to 2.
[0295] A seed culture of host strain B32 for fermentation run
060403-3 was established by adding a stock aliquot of the strain to
a 250 mL flask containing 50 mL M9-MOPS medium and antibiotics as
detailed in Table 1, and by incubating the culture overnight at
37.degree. C. on a rotary shaker at 250 rpm. The seed culture was
used to inoculate at an initial OD.sub.600 of approximately 1 a 250
mL flask containing 40 mL M9-MOPS medium and antibiotics, and the
culture was again incubated at 37.degree. C. on a rotary shaker at
250 rpm until it reached an OD.sub.600 of 3 to 5.
[0296] For all fermentation processes, the KH.sub.2PO.sub.4,
K.sub.2HPO.sub.4 3H.sub.2O, EDTA, citric acid, and
(NH.sub.4).sub.2SO.sub.4 were heat sterilized in the bioreactor (2
L Applikon Bioconsole ADI 1025s with ADI 1010 controllers, Applikon
Biotechnology, Foster City, Calif.). The remaining media components
were filter sterilized as stock solutions and injected through the
headplate. Table 3 shows the final media composition for
fermentation runs 050608-1 and 050629-1. Table 4 shows the final
media composition for fermentation run 060403-3. The starting
volume for run 050608-1 was 0.8 L, the starting volume for 050629-1
was 1.2 L and the starting volume for 060403-3 was 1 L. All runs
were inoculated by injecting 50 mL of the seed culture through the
headplate.
TABLE-US-00003 TABLE 3 Composition of Fermentation Medium of
Fermentation Runs 050608-1 and 050629-1 Component Batch Medium (per
L) Feed Solution (per L) Glucose 5 g 590-650 g KH.sub.2PO.sub.4 4.2
g -- K.sub.2HPO.sub.43H.sub.2O 15.7 g -- Citric acid 1.7 g --
(NH.sub.4).sub.2SO.sub.4 2 g -- MgSO.sub.47H.sub.2O 1.2 g 12 g EDTA
8.4 mg 13 g CoCl.sub.26H.sub.2O 0.25 mg 0.4 mg MnCl.sub.24H.sub.2O
1.5 mg 2.35 mg CuCl.sub.22H.sub.2O 0.15 mg 0.25 mg H.sub.3BO.sub.4
0.3 mg 0.5 mg Na.sub.2MoO.sub.42H.sub.2O 0.25 mg 0.4 mg
Zn(CH.sub.3COO).sub.22H.sub.2O 1.3 mg 1.6 mg Fe(III)citrate hydrate
10.0 mg 4.0 mg Thiamine HCl 4.5 mg -- Carbenicillin 100 ug 100 ug
Tetracycline 5 ug 5 ug Chloramphenicol 34 ug 34 ug
TABLE-US-00004 TABLE 4 Composition of Fermentation Medium of
Fermentation Run 060403-3 Component Batch medium (per L) Feed
solution (per L) Glucose 15 g 650 g KH.sub.2PO.sub.4 4.2 g --
K.sub.2HPO.sub.43H.sub.2O 15.7 g -- Citric acid 1.7 g --
(NH.sub.4)2SO.sub.4 2 g -- MgSO.sub.47H.sub.2O 1.2 g 12 g EDTA 8.4
mg 13 mg CoCl.sub.26H.sub.2O 2.5 mg 4 mg MnCl.sub.24H.sub.2O 15 mg
23.5 mg CuCl.sub.22H.sub.2O 1.5 mg 2.5 mg H.sub.3BO.sub.4 3 mg 5 mg
Na.sub.2MoO.sub.42H.sub.2O 2.5 mg 4 mg
Zn(CH.sub.3COO).sub.22H.sub.2O 13 mg 16 mg Fe(III)citrate hydrate
100 mg 40 mg Thiamine HCl 4.5 mg -- Carbenicillin 100 ug 100 ug
Tetracycline 5 ug 5 ug Chloramphenicol 34 ug 34 ug
[0297] For fermentation run 050608-1 (excess carbon), the feed was
initiated at induction, and feed rates were adjusted manually to
provide glucose in the concentrations shown in FIG. 12C. For
fermentation run 050629-1 (carbon-restricted), the feed was
delivered to the fermentor according to the protocol shown in Table
5. For fermentation run 060403-3 (lowest carbon), the feed was
started automatically when the initial glucose bolus (15 g) was
exhausted and the dissolved oxygen spiked. Up to a maximum of 27.6
g/hr, the rate of the feed was calculated according to the
following equation:
m.sub.s(t)=S(t.sub.0).mu.e.sup..mu.(t-t.sup.0.sup.)
.mu.=0.12
S(t.sub.0)=15 g
wherein t.sub.0 is the time at which the initial glucose was
depleted. Upon reaching the maximum rate, the glucose feed was
restricted to a rate of 9.5 g/hr, and held constant at this rate
for the remainder of the run.
TABLE-US-00005 TABLE 5 Feed Protocol for Fermentation Run 050629-1
Run Time (hours) Glucose Feed Rate (g/hr) 0 0 7 0.37 10 0.74 12
1.11 14 1.48 16 2.22 18 2.96 20 3.69 22 4.80 24 5.91 31 7.39 33
5.54 47 3.69
[0298] Runs 050608-1 and 050629-1 were carried out at 37.degree. C.
Airflow in the bioreactor was set at 1-2 L/min; pH was maintained
at 7 using ammonium hydroxide and/or sodium hydroxide; initial
agitation was 500-600 rpm; foam was controlled with antifoam B
(Sigma-Aldrich, St. Louis, Mo.); the dissolved oxygen levels were
maintained above 30% using an agitation cascade. After 5-6 hours of
cultivation, production of amorpha-4,11-diene by the host cells was
induced by adding 0.8 mL of 1 M IPTG to run 050608-1 and 1.2 mL
IPTG to run 050629-1. Upon induction, the culture temperature was
reduced to 30.degree. C.
[0299] Run 060403-3 was carried out at 30.degree. C. Airflow in the
bioreactor was set at 1-2 L/min; pH was maintained at 7 using
ammonia hydroxide. Dissolved oxygen was maintained above 30% by an
agitation cascade and oxygen enrichment. At an OD.sub.600 of
approximately 28 (19 hours after inoculation), production of
amorpha-4,11-diene by the host cells was induced by adding 1 mL 1 M
IPTG.
[0300] Amorpha-4,11-diene was captured and extracted according to
two different protocols. For runs 050608-1 and 050629-1, volatile
amorpha-4,11-diene present in the off-gas was captured by venting
the off-gas through a gas-washer containing 200 mL heptanol. The
heptanol was then diluted into ethyl acetate until the
amorpha-4,11-diene concentration in the sample was between 0.63
mg/L and 20 mg/L. For run 060403-3, amorpha-4,11-diene was captured
in the bioreactor by adding 200 mL of an organic overlay to the
fermentor at the time of induction. Product concentration was
measured by combining 25 uL broth plus organic overlay with 975 uL
acetonitrile, shaking the sample at maximum speed on a Fisher
Vortex Genie 2.TM. mixer (Scientific Industries, Inc., Bohemia,
N.Y.) for at least 3 minutes, removing cells from the sample by
centrifugation, and diluting the acetonitrile solution into ethyl
acetate until the amorpha-4,11-diene concentration in the sample
was between 0.63 and 20 mg/L. The ethyl acetate samples were
analyzed by GC/MS as described in Example 10.
[0301] As shown in FIGS. 12A and 12B, fermentation run 050608-1
(excess carbon) resulted in low maximum cell densities and low
production of amorpha-4,11-diene, respectively, correlating, at
least in part, to the relatively rapid increase in acetate levels
(FIG. 12D). In comparison, fermentation run 050629-1
(carbon-restricted) resulted in increased production of
amorpha-4,11-diene (FIG. 12B), and delayed the onset of acetate
production. These results are consistent with the hypothesis that
excess glucose feeds lead to rapid acetate production and early
cell death.
[0302] Further glucose restriction as achieved by fermentation run
060403-3 (lowest carbon) resulted in low acetate production for
over 100 hours (FIG. 12D), and significantly higher maximum cell
density and amorpha-4,11-diene production (FIGS. 12A and 12B).
Example 13
[0303] This example demonstrates increased amorpha-4,11-diene
production by an Escherichia coli host strain grown under
restricted carbon source conditions and at suboptimal
temperature.
[0304] A seed culture of host strain B153 was established by adding
a stock aliquot of the strain to a 250 mL flask containing 50 mL
M9-MOPS medium and antibiotics as detailed in Table 1, and growing
the culture at 37.degree. C. on a rotary shaker at 250 rpm to an
OD.sub.600 of 3.5 to 4.5.
[0305] 2 L bioreactors (Biocontroller ADI 1010 with Bioconsole ADI
1025, Applikon Biotechnology, Foster City, Calif.) were set up and
run in the same way as described in Example 12 for run 060403-3,
except that strain and induction time were varied.
[0306] Production of amorpha-4,11-diene in the host cells was
induced by adding 1 mL of 1 M IPTG to the culture medium. In the
fermentation run shown in FIG. 13A, amorpha-4,11-diene synthesis
was induced at an OD.sub.600 of approximately 2, while the
fermentor still contained excess glucose. In the fermentation run
shown in FIG. 13B, amorpha-4,11-diene synthesis was induced at an
OD.sub.600 of approximately 33, which was after the
glucose-restricted feed had started.
[0307] Amorpha-4,11-diene was captured and extracted according to
two different protocols. For the fermentation run shown in FIG.
13A, volatile amorpha-4,11-diene present in the off-gas was
captured by venting the off-gas through a gas-washer containing 200
mL heptanol. The heptanol was then diluted into ethyl acetate until
the amorpha-4,11-diene concentration in the sample was between 0.63
and 20 mg/L. For the fermentation run shown in FIG. 13B,
amorpha-4,11-diene was captured by adding 200 mL of an organic
overlay to the fermentor at the time of induction.
[0308] Amorpha-4,11-diene was extracted from the culture medium by
combining 25 uL broth with 975 uL acetonitrile, shaking the sample
at maximum speed on a Fisher Vortex Genie 2.TM. mixer (Scientific
Industries, Inc., Bohemia, N.Y.) for at least 3 minutes, removing
cells from the sample by centrifugation, and diluting the
acetonitrile solution into ethyl acetate until the
amorpha-4,11-diene concentration in the sample was between 0.63 and
20 mg/L. The ethyl acetate samples were analyzed by GC/MS as
described in Example 10. For the fermentation run shown in FIG.
13A, the total amount of amorpha-4,11-dien was derived by combining
the amounts present in the off-gas and in the culture medium, and
dividing the total by the fermentor volume.
[0309] The fermentation shown in FIG. 13A reached a maximal
OD.sub.600 of 93 and a maximal amorpha-4,11-diene concentration of
3.2 g/L. In contrast, the fermentation shown in FIG. 13B reached a
maximal OD.sub.600 of 245 and a maximal amorpha-4,11-diene
concentration of 15 g/L. A likely explanation for the differences
in culture growth and amorpha-4,11-diene production levels observed
in the two cultures is that in the fermentation run shown in FIG.
13A amorpha-4,11-diene production was induced before the excess
glucose was consumed, and that the unrestricted availability of
glucose caused cell death by enabling the build-up of toxic levels
of intermediates of the mevalonate pathway. In the fermentation run
shown in FIG. 13B, induction occurred after glucose delivery was
restricted, which prevented the build-up of pathway intermediates,
leading to higher cell density and amorpha-4,11-diene production
levels.
Example 14
[0310] This example demonstrates increased amorpha-4,11-diene
production by an Escherichia coli host strain grown under
restricted carbon and nitrogen source conditions and at suboptimal
temperature.
[0311] A seed culture of host strain B86 was established by adding
a stock aliquot of the strain to a 250 mL flask containing 50 mL
M9-MOPS medium and antibiotics as detailed in Table 1. The culture
was grown overnight at 37.degree. C. on a rotary shaker at 250 rpm,
sub-cultured the following morning into the same medium at an
OD.sub.600 of approximately 1, and grown again at 37.degree. C. and
250 rpm to an OD.sub.600 of 3 to 5.
[0312] Four 2 L bioreactors (Biocontroller ADI 1010 with Bioconsole
ADI 1025, Applikon Biotechnology, Foster City, Calif.) were set up
and run in the same way as described in Example 12 for run
060403-3, except that the nitrogen restricted runs did not contain
ammonia sulfate in the feed.
[0313] An exponential glucose feed with a 6 hour doubling time was
initiated automatically when the initial glucose bolus (15 g) was
exhausted and the dissolved oxygen spiked. Up to a maximum of 30.4
g/hr, the rate of the feed was calculated according to the
following equation:
m.sub.s(t)=S.sub.0.mu.e.sup..mu.(t-t.sup.0.sup.)
.mu.=0.12 min.sup.-1
S.sub.0=15 g
wherein .mu. is the specific growth rate, and t.sub.0 is the time
at which the initial glucose bolus was depleted. Upon reaching the
maximum rate, the glucose feed was reduced to a rate of 11.4 g/hr,
and held constant at this rate for the remainder of the run. In
fermentation runs 060710-4, 060724-5, and 060619-5 (carbon- and
nitrogen-restricted), the glucose feed was further reduced when
ammonia restriction lead to glucose accumulation in the medium.
[0314] Fermentation was carried out at the reduced temperature of
30.degree. C. Airflow in the bioreactor was set at 1 vvm; initial
agitation was at 700 rpm; foam was controlled with antifoam B
(Sigma-Aldrich, St. Louis, Mo.); and dissolved oxygen tension was
controlled at 40% using an agitation cascade (700-1,200 rpm) and
oxygen enrichment. In fermentation run 060327-3
(carbon-restricted), the pH was maintained at 7 using 20%
NH.sub.4OH; in fermentation runs 060710-4, 060724-5, and 060619-5
(carbon- and nitrogen-restricted), pH was maintained at 7 initially
using 20% NH.sub.4OH, and starting at 72 hours using a 50/50
mixture of 2.5 N NaOH and 10 N NH.sub.4OH, to further restrict the
amount of ammonia going into the fermentor.
[0315] Production of amorpha-4,11-diene in the host cells was
induced at an OD.sub.600 of approximately 30 by adding 1 mL of 1 M
IPTG to the culture medium.
[0316] Amorpha-4,11-diene was captured by overlaying the medium
with 10% (v/v) of an organic overlay. Amorpha-4,11-diene was then
extracted by combining 25 uL of broth with 975 uL methanol, shaking
the sample at maximum speed on a Fisher Vortex Genie2.TM. mixer
(Scientific Industries, Inc., Bohemia, N.Y.) for at least 15
minutes, removing cells from the sample by centrifugation, and
adding 10 uL of the methanol solution to 990 uL ethyl acetate
containing 10 uL/L trans-caryophylene.
[0317] Samples were analyzed by GC/MS as described in Example
10.
[0318] FIGS. 14A-E show data from fermentation run 060327-3
(carbon-restricted). The fermentation produced a maximum
concentration of amorpha-4,11-diene of 16 g/L (FIG. 14A). The
maximum volumetric productivity of the host strain was more than
200 mg/L/hour (FIG. 14B). The maximum specific productivity of the
host strain was >2 mg/L/hour/OD.sub.600 (FIG. 14C). The
concentration of ammonia in the culture medium was about 30 mM at
the start of the fermentation run, rose to about 76 mM upon
addition of the feed solution during the exponential growth phase,
and remained above 60 mM for the remainder of the run (FIG. 14D).
The maximum OD.sub.600 reached was about 290 (FIG. 14D),
corresponding to 116 g DCW/L. The concentration of glucose in the
culture medium dropped from 15 g/L to below 1 g/L in less than 20
hours, and remained low (FIG. 14E). Acetate levels were low
throughout the fermentation (FIG. 14E).
[0319] FIGS. 15A-E show data from fermentation runs 060710-4,
060724-5, and 060619-5 (carbon- and nitrogen-restricted). The
fermentations produced a maximum concentration of
amorpha-4,11-diene from about 20 g/L to 30 g/L (FIG. 15A). The
maximum volumetric productivity of the host strain was more than
400 mg/L/hour in all three fermentation runs (FIG. 15B), which is
significantly higher than the maximum volumetric productivity
obtained in the nitrogen unrestricted fermentation (FIG. 14B). The
maximum specific productivity of the host strain was >2
mg/L/hour/OD.sub.600 for all runs, and remained high throughout the
runs (FIG. 15C). The concentration of ammonia in the culture medium
was about 35 mM to 50 mM at the start of the fermentation runs,
dropped upon addition of the feed solution during exponential
growth, and remained below 10 mM for the remainder of the run (FIG.
15D). (The lowered ammonia levels compared to fermentation run
060327-3 (FIG. 14D) are due to the lack of ammonia in the feed
solution and reduced ammonia in the base used to maintain the pH.
Fermentation runs 060710-4 and 060619-5 showed a spike in ammonia
concentration at the end of the runs, but the spikes occurred after
the bulk of the production of amorpha-4,11-diene.) The maximum
OD.sub.600 reached was 170 to 220 (FIG. 15D), corresponding to 68 g
to 88 g DCW/L. The concentration of glucose in the culture medium
dropped from 15 g/L to below 1 g/L in less than 20 hours, and
remained low (FIG. 15E). Acetate levels were low throughout the
fermentation runs (FIG. 15E).
Example 15
[0320] This example describes the production of amorpha-4,11-diene
via the DXP pathway in an Escherichia coli host strain.
[0321] Seed cultures of host strains B003, B617, B618, and B619
were established by adding a stock aliquot of each strain to
separate 125 mL flasks containing 25 mL M9-MOPS and antibiotics as
detailed in Table 1, and by growing the cultures overnight.
[0322] The seed cultures were used to inoculate at an initial
OD.sub.600 of approximately 0.05, separate 250 mL flasks containing
40 mL M9-MOPS medium, 45 ug/mL thiamine, micronutrients, 1.00E-5
mol/L FeSO4, 0.1 M MOPS, 0.5% yeast extract, 20 g/L of D-glucose,
and antibiotics. Cultures were incubated at 30.degree. C. in a
humidified incubating shaker at 250 rpm until they reached an
OD.sub.600 of 0.2 to 0.3, at which point the production of
amorpha-4,11-diene in the host cells was induced by adding 40 uL of
1M IPTG to the culture medium.
[0323] At the time of induction, the cultures were overlain with 8
mL of an organic overlay to capture the amorpha-4,11-diene. Samples
were taken at various time points, and amorpha-4,11-diene was
extracted and analyzed by GC/MS as described in Example 10.
Experiments were performed using 2 independent clones of each host
strain, and results were averaged. Deviation between samples was
found to be less than 10%.
[0324] As shown in FIG. 16, Escherichia coli host strain B619,
which comprises nucleotide sequences encoding enzymes of the full
engineered DXP pathway, produced approximately 45 mg/g DCW
amorpha-4,11-diene.
Example 16
[0325] This example describes the production of
3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol in Escherichia
coli host strains.
[0326] Seed cultures of host strains B286, B287, B288, and B291
were established by streaking out a stock aliquot of each strain on
LB agar containing antibiotics as detailed in Table 1. Three
independent colonies were picked for each strain, and each colony
was inoculated into 7 mL of LB media containing antibiotics. The
cultures were grown overnight at 37.degree. C. on a rotary shaker
at 250 rpm until late exponential phase. The cultures were then
inoculated at an OD.sub.600 of approximately 0.05, into a 250 mL
flask containing 40 ml of M9-MOPS, 2% glucose, 0.5% yeast extract,
and antibiotics. The cultures were grown overnight at 37.degree. C.
on a rotary shaker at 250 rpm until they reached an OD.sub.600 of
approximately 0.2, at which point they were induced by adding 40 uL
of 1 M IPTG. The cultures were grown for 72 hours at 30.degree. C.
on a rotary shaker at 250 rpm. One to two times per day, the
OD.sub.600 of each culture was measured, and a 700 uL sample was
removed. To extract the 3-methyl-but-3-en-1-ol and
3-methyl-but-2-en-1-ol from the culture broth, 600 uL of ethyl
acetate was added to 300 uL of each removed sample. The sample was
then vortexed for 15 minutes, and 400 uL of the upper ethyl acetate
phase was transferred to a clean glass vial for analysis.
[0327] The samples were analyzed on a Hewlett-Packard 6890 gas
chromatograph/mass spectrometer (GC/MS). A 1 uL sample was
separated on the GC using a DB-5 column (Agilent Technologies,
Inc., Palo Alto, Calif.) and helium carrier gas. The temperature
program for the analysis was as follows: 60.degree. C. for 3
minutes, increasing temperature at 60.degree. C./minute to a
temperature of 300.degree. C., and a hold at 300.degree. C. for 2
minutes. The total run time was 9 minutes. The resolved samples
were analyzed by a Hewlett-Packard model 5973 mass selective
detector. Previous mass spectra demonstrated that
3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol have a retention
time of 2.067 minutes using this GC protocol. To focus detection on
3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol, a
selective-ion-monitoring method was employed that monitors only
ions 56 and 68 in 3-methyl-but-3-en-1-ol and
3-methyl-but-2-en-1-ol.
Example 17
[0328] This example describes the production of amorpha-4,11-diene
by a Saccharomyces cerevisiae host strain.
[0329] The generation of host strain EPY224 is described in Ro et
al. (Nature 440: 940-943; 2006) and in PCT Patent Publication
WO2007/005604. Host strain EPY224 was cured of expression plasmid
pRS425ADS by growth in YPD medium (Methods in Yeast Genetics: A
Cold Spring Harbor Laboratory Course Manual, 2005 ed., ISBN
0-87969-728-8), plating for single colonies on YPD agar, and then
patching single colonies onto CSM-Met His agar and CSM-Met Leu
agar. Clones that grew on CSM-Met His agar but not on CSM-Met Leu
agar were cured (i.e., had lost the plasmid pRS425ADS). One such
clone was designated EPY300. EPY300 was transformed with expression
plasmid pRS425-ADS-LEU2d, a plasmid identical to pRS425-ADS except
that instead of LEU2 it contains a LEU2d selection marker (Erhart
and Hollenberg (1983) J. Bacteriol. 156: 625-635) yielding host
strain Y185.
[0330] Y185 host cell transformants were selected on synthetic
defined media, containing 2% glucose and all amino acids except
histidine, leucine, and methionine (CSM-glucose; MP Biomedicals,
Solon, Ohio). The host strain EPY300 is auxotrophic for leucine
biosynthesis (leu2), but expression plasmid pRS425-ADS-LEU2d in
Y185 restores leucine prototrophy (LEU2). Single colonies were
patched onto selective medium (CSM-glucose-histidine, leucine,
methionine), and grown for 2 days. The cells were scraped from the
plate and transferred to 1 mL of 25% (v/v) glycerol in a cryotube.
The suspension was mixed, and then stored at -80.degree. C.
[0331] Seed flasks of host strain Y185 were established by adding a
stock aliquot of the strain to a 125 mL flask containing 25 mL of
CSM-glucose lacking leucine and methionine, and by growing the
cultures overnight. The cultures were used to inoculate at an
initial OD.sub.600 of approximately 0.05 a 250 mL baffled flask
containing 40 mL of synthetic defined media lacking leucine, and
containing 0.2% glucose, 1.8% galactose, and 1 mM methionine. The
culture was incubated at 30.degree. C. on a rotary shaker at 200
rpm. Because the presence of glucose in the media prevents
induction of the GAL1 promoter by galactose, amorpha-4,11-diene
production was not induced until the cells had used up the glucose
in the media and had switched to using galactose as their main
carbon source. At the time of inoculation, the cultures were
overlain with 8 mL of an organic overlay to capture the
amorpha-4,11-diene. Samples were taken at 72 hours by transferring
5 uL of the organic solvent layer to a clean glass vial containing
500 uL ethyl acetate containing a known concentration of beta- or
trans-caryophyllene as an internal standard.
[0332] The organic overlay/ethyl acetate samples were analyzed on a
Hewlett-Packard 6890 gas chromatograph/mass spectrometer (GC/MS) as
described in Example 10.
[0333] After 72 hours of growth, 3 yeast cultures were found to
produce 60.68, 54.48, and 59.25 mg/L amorpha-4,11-diene.
Example 18
[0334] This example describes the production of amorpha-4,11-diene
in an Saccharomyces cerevisiae host strain where the host strain
includes a native mevalonate pathway as well as a heterologous
mevalonate pathway that is under control of a heterologous
regulatory control.
[0335] Yeast strains CEN.PK2-1C (Y002) (MATA; ura3-52; trp1-289;
leu2-3,112; his3.DELTA.1; MAL2-8C; SUC2) and CEN.PK2-1D (Y003)
(MATalpha; ura3-52; trp1-289; leu2-3,112; his3.DELTA.1; MAL2-8C;
SUC2) (J. P. van Dijken et al., Enzyme Microb Technol 26, 706 (Jun.
1, 2000) were cultivated in either standard rich medium (YPD) or in
defined synthetic medium (D. Rose, F. Winston, P. Heiter, Methods
in yeast genetics: a laboratory course manual. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1990) lacking
appropriate nutrients allowing for selection of integrative
transformants, plasmid retention, and meiotic progeny.
[0336] DNA-mediated transformations into S. cerevisiae were
conducted using the lithium acetate procedure as described by R. H.
Schiestl, R. D. Gietz, Curr Genet. 16, 339 (December, 1989). All
gene disruptions and replacements were confirmed by phenotypic
analysis, colony polymerase chain reaction ("PCR") and sequencing
of amplified genomic DNA. Plasmids pAM489-pAM498 were constructed
using the pCR 2.1 (Invitrogen, Carlsbad Calif.) and are
schematically described by FIG. 7A-C and Table 6. The HISMX marker
sequences are described in M. S. Longtine et al., Yeast 14, 953
(July, 1998). Propagation of plasmid DNA was performed in
Escherichia coli strain DH5.alpha..
TABLE-US-00006 TABLE 6 Crick Watson Strain 5'HR Gene #1 Promoter
Promoter Gene #2 Genetic Marker 3'HR pAM489 TRP1 tHMGR GAL1 GAL10
ERG20 TRP1 TRP1 pAM490 TRP1 tHMGR CUP1 CUP1 ERG20 TRP1 TRP1 pAN491
URA3 tHMGR GAL1 GAL10 ERG13 URA3 URA3 pAM492 URA3 IDI1 CUP1 CUP1
tHMGR URA3 URA3 pAM493 ADE1 tHMGR GAL1 GAL10 IDI1 ADE1 URA3 pAM494
ADE1 tHMGR CUP1 CUP1 IDI1 ADE1 ADE1 pAM495 HIS3 ERG12 GAL1 GAL10
ERG10 HISMX HIS3 pAM496 HIS3 ERG12 CUP1 CUP1 ERG10 HISMX HIS3
pAM497 LEU2 ERG19 GAL1 GAL1 ERG8 HISMX LEU2 pAM498 LEU2 ERG19 CUP1
CUP1 ERG8 HISMX LEU2
[0337] S. cerevisiae strains Y002 and Y003 were prepared for
introduction of inducible mevalonate pathway genes by the
following. The ERG9 promoter was replaced with the S. cerevisiae
MET3 promoter by PCR amplification of the KanMX-PMET3 region from
pAM328 (SEQ ID NO: 43) using primers 50-56-pw100-G (SEQ ID NO: 44)
and 50-56-pw101-G (SEQ ID NO: 45) containing 45 basepairs of
homology to the native ERG9 promoter. 10 .mu.g of the resulting PCR
product was transformed into exponentially growing Y002 and Y003
strains using 40% w/w polyethelene glycol 3350 (Sigma-Aldrich St
Louis, Mo.), 100 mM lithium acetate (Sigma), 10 .mu.g Salmon Sperm
DNA (Invitrogen) and incubation at 30.degree. C. for 30 minutes
followed by a 42.degree. C. heat shock for 30 minutes (as described
by Schiestl & Gietz, Curr. Genet. 16: 339 (1989)). Positive
recombinants were identified by their ability to grow on rich
medium containing 0.5 .mu.g/ml Geneticin (Invitrogen Co, Carlsbad,
Calif.) and confirmed by diagnostic PCR. The resultant clones were
given the designation Y93 (MAT A) and Y94 (MAT alpha). Next, the
ADE1 open reading frame was replaced with the Candida glabrata LEU2
gene (CgLEU2). The 3.5 KB CgLEU2 genomic locus was amplified from
C. glabrata genomic DNA (ATCC, Manassas, Va.) using primers
61-67-CPK066-G (SEQ ID NO: 46) and 61-67-CPK067-G (SEQ ID NO: 47)
containing 50 basepairs of flanking homology to the ADE1 open
reading frame (ORF). 10 .mu.g of the resulting PCR product was
transformed into exponentially growing Y93 and Y94 as described
above. ade1-strains were selected for growth in the absence of
leucine supplementation and confirmed by diagnostic PCR. The
resultant clones were given the designation Y176 (MAT A) and Y177
(MAT alpha).
[0338] To generate S. cerevisiae strain Y188, 2 .mu.g's of plasmid
DNA from pAM491 (SEQ ID NO: 48) and pAM495 (SEQ ID NO:49),
respectively, were digested overnight with PmeI (New England
Biolabs, Beverly, Mass.) and introduced into exponentially growing
Y176 as described above. Positive recombinants were selected for by
growth on medium lacking uracil and histidine. Integration into the
correct genomic locus was confirmed by diagnostic PCR.
[0339] To generate S. cerevisiae strain Y189, 2 .mu.g's of plasmid
DNA from pAM489 (SEQ ID NO: 50) and pAM497 (SEQ ID NO: 51),
respectively, were digested overnight with PmeI and introduced into
exponentially growing Y177 as described above. Positive
recombinants were selected for by growth on medium lacking
tryptophan and histidine. Integration into the correct genomic
locus was confirmed by diagnostic PCR.
[0340] Approximately 1.times.10.sup.7 cells from Y188 and Y189 were
mixed on a YPD medium plate for 6 hours at room temperature to
allow for mating. The mixed cell culture was then plated to medium
lacking histidine, uracil and tryptophan to select for growth of
diploid cells. 2 .mu.g of plasmid DNA from pAM493 (SEQ ID NO: 52)
was digested overnight with PmeI and introduced into exponentially
growing diploid cells as described above. Positive recombinants
were selected for by growth on medium lacking adenine. Integration
into the correct genomic locus was confirmed by diagnostic PCR. The
resultant strain was given the designation Y238.
[0341] To generate haploid strains containing the full complement
of introduced genes, Y238 was sporulated in 2% potassium acetate
and 0.02% raffinose liquid medium. Approximately 200 genetic
tetrads (tetrads are four-spored meiotic products) were isolated
using a Singer Instruments MSM300 series micromanipulator (Singer
Instrument Co, LTD. Somerset, UK). Independent genetic isolates
containing the appropriate complement of introduced genetic
material were identified by their ability to grow in the absence of
adenine, histidine, uracil, and tryptophan. Integration of all
introduced DNA was confirmed by diagnostic PCR. The resultant
strains were given the designation Y210 (MAT A) and Y211 (MAT
alpha).
[0342] 2 .mu.g of plasmid DNA from pAM426 (SEQ ID NO:53),
containing S. cerevisiae condon optimized Amorphadeine Synthase
(ADS) expressed from the S. cerevisiae GAL1 promoter, was
introduced into exponentially growing Y210 and Y211 as described
above. S. cerevisiae strains that contained the pAM426 plasmid were
selected for by their ability to grow in the absence of leucine
supplementation. The resultant strains were given the designation
Y225 (MAT A) and Y227 (MAT alpha).
[0343] 2 .mu.g of plasmid DNA from pAM322 (SEQ ID NO: 54),
containing S. cerevisiae condon optimized Amorphadeine Synthase
(ADS) and cytochrome P450 monooxygenase (AMO) expressed from the S.
cerevisiae GAL1 and the cytochrome P450 oxidoreductase (CPR)
expressed from the S. cerevisiae GAL10 promoter, was introduced
into exponentially growing Y210 and Y211 as described above. S.
cerevisiae strains that contained the pAM322 plasmid were selected
for by their ability to grow in the absence of leucine
supplementation. The resultant strains were given the designation
Y222 (MAT A) and Y224 (MAT alpha).
Example 19
[0344] This example describes the production of .alpha.-farnesene
or .beta.-farnesene in Escherichia coli host strains.
[0345] Seed cultures of host strains B552 and B592 were established
by adding a stock aliquot of each strain to a 125 mL flask
containing 25 mL M9-MOPS, 0.8% glucose, 0.5% yeast extract, and
antibiotics as detailed in Table 1, and by growing the cultures
overnight.
[0346] The seed cultures were used to inoculate at an initial
OD.sub.600 of approximately 0.05, 250 mL flasks containing 40 mL
M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics. Cultures
were incubated at 30.degree. C. on a rotary shaker at 250 rpm until
they reached an OD.sub.600 of approximately 0.2, at which point the
production of .alpha.-farnesene or .beta.-farnesene in the host
cells was induced by adding 40 uL of 1 M IPTG. At the time of
induction, the cultures were overlain with 8 mL of an organic
overlay to capture the .alpha.-farnesene. Samples were taken every
24 hours up to 120 hours (total of 5 time points) by transferring 2
uL to 10 uL of the organic overlay layer to a clean glass vial
containing 1 mL ethyl acetate spiked with trans-caryophyllene as an
internal standard. In addition, 1 mL aliquots of the cultures were
spun down, cell pellets were resuspended in 250 uL sterile water,
and the cell suspensions were transferred to a glass vial
containing 1 mL ethyl acetate spiked with trans-caryophyllene as an
internal standard. In addition, 0.5 mL aliquots of the whole
culture broth were added to a glass vials containing 1 mL ethyl
acetate spiked with trans-caryophyllene as an internal standard.
The whole culture broth samples were extracted in the ethyl acetate
by vortexing the glass vials for 10 minutes, after which 600 uL of
the ethyl acetate extraction was transferred to a clean glass
vial.
[0347] The organic overlay/ethyl acetate samples and the ethyl
acetate-extracted whole culture broth samples were analyzed on an
Agilent 6890N gas chromatograph equipped with an Agilent 5975 mass
spectrometer (GC/MS) in full scan mode (50-500 m/z). To expedite
run times, the temperature program and column matrix was modified
to achieve optimal peak resolution and the shortest overall
runtime. A 1 uL sample was separated using a HP-5MS column (Agilent
Technologies, Inc., Palo Alto, Calif.) and helium carrier gas. The
temperature program for the analysis was as follows: 150.degree. C.
hold for 3 minutes, increasing temperature at 25 C/minute to a
temperature of 200.degree. C., increasing temperature at 60.degree.
C./minute to a temperature of 300.degree. C., and a hold at
300.degree. C. for 1 minute. Previous mass spectra demonstrated
that the .beta.-farnesene synthase product was .beta.-farnesene,
and that .beta.-farnesene had a retention time of 4.33 minutes
using this GC protocol. Farnesene titers were calculated by
comparing generated peak areas against a quantitative calibration
curve of purified .beta.-farnesene (Sigma-Aldrich Chemical Company,
St. Louis, Mo.) in trans-caryophyllene-spiked ethyl acetate.
[0348] Host strain B592 produced approximately 400 mg/L of
.alpha.-farnesene at 120 hours (averaged over 3 independent
clones), and had a maximal specific productivity of approximately
46 mg/L/OD.sub.600. Host strain B552 produced approximately 1.1 g/L
of .beta.-farnesene at 120 hours (averaged over 3 independent
clones), and had a maximal specific productivity of approximately
96 mg/L/OD.sub.600 (1 representative clone).
Example 20
[0349] This example describes the production of .beta.-farnesene
via the DXP pathway in an Escherichia coli host strain.
[0350] Seed cultures of host strains B650, B651, B652, and B653
were established by adding a stock aliquot of each strain to
separate 125 mL flasks containing 25 mL M9-MOPS and antibiotics as
detailed in Table 1, and by growing the cultures overnight.
[0351] The seed cultures were used to inoculate at an initial
OD.sub.600 of approximately 0.05 separate 250 mL flasks containing
40 mL M9-MOPS minimal medium, 45 ug/mL thiamine, micronutrients,
1.00E-5 mol/L FeSO4, 0.1 M MOPS, 0.5% yeast extract, 20 g/L of
D-glucose, and antibiotics. The cultures were incubated at
30.degree. C. in a humidified incubating shaker at 250 rpm until
they reached an OD.sub.600 of 0.2 to 0.3, at which point the
production of .beta.-farnesene in the host cells was induced by
adding 40 uL of 1 M IPTG to the culture medium. At the time of
induction, the cultures were overlain with 8 mL of an organic
overlay to capture the .beta.-farnesene. Samples were taken at
various time points by transferring 100 uL samples of the upper
organic overlay layer to a clean tube. The tube was centrifuged to
separate out any remaining cells or media, and 10 uL of the organic
overlay samples were transferred into 500 uL ethyl acetate spiked
with beta- or trans-caryophyllene as an internal standard in clean
glass GC vials. The mixtures were vortexed for 30 seconds, and then
analyzed as described in Example 18. Escherichia coli host strain
B653 produced approximately 7 mg/g DCW .beta.-farnesene.
Example 21
[0352] This example describes the production of .alpha.-farnesene
or .beta.-farnesene in a Saccharomyces cerevisiae host strain.
[0353] Strain EPY300 was generated by removing the expression
plasmid from Saccharomyces cerevisiae strain EPY224 (Ro et al.
(2006) Nature 440: 940-943; PCT Patent Publication WO2007/005604)
by culturing in rich medium. Strain EPY300 was then transformed
with expression plasmids pRS425-FSA or pR425-FSB, yielding host
strains Y166 and Y164, respectively.
[0354] Host cell transformants were selected on synthetic defined
media, containing 2% glucose and all amino acids except leucine
(SM-glu). The host strain EPY300 was auxotrophic for leucine
biosynthesis (leu2), but expression plasmid pRS425-FSA or
pRS425-FSB restores leucine prototrophy (LEU2). Single colonies
were transferred to culture vials containing 5 mL of liquid SM-glu
lacking leucine. The cultures were incubated by shaking at
30.degree. C. until growth reaches stationary phase. The cells were
stored at -80.degree. C. in cryo-vials in 1 mL frozen aliquots made
up of 400 .mu.L 50% glycerol and 600 .mu.L liquid culture.
[0355] Seed cultures were established by adding a stock aliquot to
a 125 mL flask containing 25 mL SM-glu lacking leucine, and growing
the cultures overnight. The seed cultures were used to inoculate at
an initial OD.sub.600 of approximately 0.05 250 mL baffled flasks
containing 40 mL of synthetic defined media lacking leucine, 0.2%
glucose, and 1.8% galactose. Cultures were incubated at 30.degree.
C. on a rotary shaker at 200 rpm. Because the presence of glucose
in the media prevents induction of the Gal1 promoter by galactose,
farnesene production was not induced until the cells use up the
glucose in the media and switch to using galactose as their main
carbon source. The cultures are overlain with 8 mL methyl oleate or
isopropyl myristate. Samples were taken once every 24 hours by
transferring 2-10 uL of the organic solvent layer to a clean glass
vial containing 500 uL ethyl acetate containing a known
concentration of beta- or trans-caryophyllene as an internal
standard. In addition, 0.5 mL aliquots of the whole culture broth
were added to a glass vials containing 1 mL ethyl acetate spiked
with trans-caryophyllene as an internal standard. The whole culture
broth samples were extracted in the ethyl acetate by vortexing the
glass vials for 10 minutes, after which 600 uL of the ethyl acetate
extraction was transferred to a clean glass vial.
[0356] Host strain Y166 produced approximately 9.8 mg/L of
.alpha.-farnesene at 120 hours (averaged over 3 independent
clones), and had a maximal specific productivity of approximately 3
mg/L/OD.sub.600 (1 representative clone). Host strain Y164 produced
approximately 56 mg/L of .beta.-farnesene at 120 hours (averaged
over 3 independent clones), and had a maximal specific productivity
of approximately 20 mg/L/OD.sub.600 (1 representative clone).
Example 22
[0357] This example describes the production of .gamma.-terpinene,
.alpha.-pinene, and terpinolene in Escherichia coli host
strains.
[0358] Seed cultures of host strains for production of
.gamma.-terpinene (E. coli DH1-T1r [pMevT, pMevB-Gpps, pAM445]),
.alpha.-pinene (E. coli DH1-T1r [pMevT, pMevB-Gpps, pAM443 or
pAM442]) or terpinolene (E. coli DH1-T1r [pMevT, pMevB-Gpps,
pAM444] were established by adding a stock aliquot of each strain
to separate 125 mL flasks containing 25 mL M9-MOPS, 2% glucose,
0.5% yeast extract, and antibiotics as detailed in Table 1, and by
growing the cultures overnight to late exponential phase.
[0359] The seed cultures were used to inoculate at an initial
OD.sub.600 of approximately 0.05, 250 mL flasks containing 40 mL
M9-MOPS, 2% glucose, 0.5% yeast extract, and antibiotics. At time
of inoculation, the cultures were also overlain with 4 mL
hexadecane. Cultures were incubated at 30.degree. C. on a rotary
shaker at 200-250 rpm until they reached an OD.sub.600 of
approximately 0.2, at which point the production of the compound of
interest in the host cells in the host cells was induced by adding
40 uL of 1 M IPTG. Samples were taken once per day for 96 hours by
transferring 200 uL of the hexadecane layer to a 0.6 mL microfuge
tube. For analysis, the hexadecane overlay was diluted 1:1 or 1:10
with ethyl acetate spiked with trans-caryophyllene as an internal
standard in a 1.8 mL GC vial. In addition, 1 mL aliquots of the
cultures were spun down, cell pellets were resuspended in 250 uL
sterile water, and the cell suspensions were transferred to a glass
vial containing 1 mL ethyl acetate spiked with trans-caryophyllene
as an internal standard. The cell pellets were extracted in the
ethyl acetate by vortexing the glass vials for 15 minutes, after
which 500 uL of the ethyl acetate extraction was transferred to a
clean glass vial.
[0360] The hexadecane/ethyl acetate samples and the ethyl
acetate-extracted cell pellet samples were analyzed on an Agilent
6890N gas chromatograph equipped with an Agilent 5975 mass
spectrometer (GC/MS) in full scan mode (50-500 m/z). To expedite
run times, the temperature program and column matrix was modified
to achieve optimal peak resolution and the shortest overall
runtime. A 1 .mu.L sample was split (a split ratio between 1:2 and
1:50 was selected based on sample concentration) and then separated
using a HP-5MS column (Agilent Technologies, Inc., Palo Alto,
Calif.) and helium carrier gas. The temperature program for the
analysis was as follows: 75.degree. C. hold for 3 minutes,
increasing temperature at 20.degree. C./minute to a temperature of
115.degree. C., increasing temperature at 60.degree. C./minute to a
temperature of 300.degree. C., and a hold at 300.degree. C. for 0.5
minute. The various products, .gamma.-terpinene, .alpha.-pinene,
and terpinolene were observed at 5.4, 4.1, 5.4, and 5.9 minutes,
respectively. Titers were calculated by comparing generated peak
areas against a quantitative calibration curve of purified
standards in trans-caryophyllene-spiked ethyl acetate.
Example 23
[0361] This example describes the production of linalool, limonene,
.beta.-pinene, .beta.-phellandrene, carene, or sabinine in
Escherichia coli host strains.
[0362] Seed cultures are established by adding a stock aliquot of
each strain to separate 125 mL flasks containing 25 mL M9-MOPS,
0.5% yeast extract, 2% glucose, and antibiotics as detailed in
Table 1, and by growing the cultures overnight.
[0363] The seed cultures are used to inoculate at an initial
OD.sub.600 of approximately 0.05, 250 mL baffled flasks containing
40 mL M9-MOPS, 0.5% yeast extract, 2% glucose, and antibiotics.
Cultures are incubated at 30.degree. C. on a rotary shaker at 250
rpm until they reach an OD.sub.600 of approximately 0.2, at which
point the production of the compound of interest in the host cells
is induced by adding 40 ul of 1 M IPTG to the culture medium. The
compound of interest is separated from the culture medium through
solvent-solvent extraction, or by settling and decantation if the
titer of the compound of interest is large enough to saturate the
media and to form a second phase.
Sequence CWU 1
1
5414247DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1gaattcaaag gaggaaaata aaatgaagaa
ctgtgtgatt gtttctgcgg tccgcacggc 60gatcggcagc tttaacggct ctttagcgag
cacctctgca atcgatctgg gtgcgacggt 120cattaaggcc gccattgaac
gcgccaaaat cgacagccag cacgttgatg aggtgatcat 180gggcaatgtg
ttacaagccg gcctgggtca aaacccagcg cgtcaagcac tgttaaaatc
240tggtctggcc gagaccgtgt gtggcttcac cgtcaataag gtttgcggct
ctggcctgaa 300gagcgtggcc ctggcagcac aagcgattca agccggtcag
gcacaaagca tcgttgcggg 360tggcatggag aacatgtctc tggcgccgta
cttattagat gccaaagccc gcagcggtta 420tcgcctgggc gatggtcagg
tgtacgacgt catcttacgc gatggcttaa tgtgcgcgac 480ccacggttac
cacatgggta ttacggccga aaacgtggcg aaagaatacg gcattacgcg
540cgagatgcag gatgaattag cactgcactc tcagcgcaaa gcagcagccg
cgatcgagtc 600tggtgcgttt acggcggaaa tcgtgccagt taacgtggtc
acgcgcaaga agacgttcgt 660tttcagccag gacgagttcc cgaaggcaaa
cagcaccgcg gaggccttag gtgccttacg 720cccagccttt gacaaagcgg
gcacggtcac cgccggtaat gcgagcggca tcaatgatgg 780tgcagcggca
ctggtcatca tggaagagag cgccgcatta gcagcgggtc tgaccccatt
840agcgcgcatt aaatcttatg ccagcggcgg cgtcccacca gccctgatgg
gcatgggtcc 900ggtcccagcc acgcaaaaag ccctgcaatt agcgggcctg
caactggccg acattgatct 960gatcgaggcg aacgaggcgt ttgcagcgca
gttcctggcg gtgggtaaga atctgggctt 1020cgacagcgag aaagtcaatg
tgaacggtgg cgcgattgcg ttaggccatc cgattggtgc 1080aagcggcgca
cgcatcttag tgacgttact gcacgccatg caggcacgcg acaagacctt
1140aggcctggcg accttatgta ttggtggcgg tcaaggtatc gccatggtga
tcgaacgcct 1200gaactgaaga tctaggagga aagcaaaatg aaactgagca
ccaagctgtg ctggtgtggc 1260atcaagggtc gcctgcgccc acaaaagcag
caacagctgc acaacacgaa cctgcaaatg 1320accgagctga aaaagcagaa
gacggccgag caaaagaccc gcccgcagaa cgttggcatc 1380aagggcatcc
agatttatat cccgacgcag tgtgtcaacc aatctgagct ggagaaattc
1440gatggcgtca gccagggtaa gtacaccatc ggcctgggcc agaccaacat
gagcttcgtg 1500aacgaccgtg aggacatcta ttctatgagc ctgacggtgc
tgtctaagct gatcaagagc 1560tacaacatcg acacgaataa gatcggtcgt
ctggaggtgg gtacggagac gctgattgac 1620aagagcaaaa gcgtgaagtc
tgtcttaatg cagctgttcg gcgagaacac ggatgtcgag 1680ggtatcgaca
ccctgaacgc gtgttacggc ggcaccaacg cactgttcaa tagcctgaac
1740tggattgaga gcaacgcctg ggatggccgc gatgcgatcg tcgtgtgcgg
cgatatcgcc 1800atctatgaca agggtgcggc acgtccgacc ggcggtgcag
gcaccgttgc gatgtggatt 1860ggcccggacg caccaattgt cttcgattct
gtccgcgcgt cttacatgga gcacgcctac 1920gacttttaca agccggactt
cacgagcgaa tacccgtacg tggacggcca cttctctctg 1980acctgctatg
tgaaggcgct ggaccaggtt tataagtctt atagcaaaaa ggcgatttct
2040aagggcctgg tcagcgaccc ggcaggcagc gacgccctga acgtgctgaa
gtatttcgac 2100tacaacgtgt tccatgtccc gacctgcaaa ttagtgacca
aatcttatgg ccgcctgtta 2160tataatgatt tccgtgccaa cccgcagctg
ttcccggagg ttgacgccga gctggcgacg 2220cgtgattacg acgagagcct
gaccgacaag aacatcgaga agaccttcgt caacgtcgcg 2280aagccgttcc
acaaagagcg tgtggcccaa agcctgatcg tcccgaccaa cacgggcaac
2340atgtataccg cgtctgtcta cgcggcattc gcgagcctgc tgaattacgt
cggttctgac 2400gacctgcagg gcaagcgcgt tggcctgttc agctacggta
gcggcttagc ggccagcctg 2460tatagctgca aaattgtcgg cgacgtccag
cacatcatca aggagctgga catcaccaac 2520aagctggcga agcgcatcac
cgagacgccg aaagattacg aggcagcgat cgagttacgc 2580gagaatgcgc
atctgaagaa gaacttcaag ccgcaaggta gcatcgagca cctgcagagc
2640ggcgtctact acctgacgaa cattgacgac aagttccgcc gttcttatga
cgtcaaaaag 2700taactagtag gaggaaaaca tcatggtgct gacgaacaaa
accgtcatta gcggcagcaa 2760ggtgaagtct ctgagcagcg cccaaagctc
tagcagcggc ccgtctagca gcagcgagga 2820ggacgacagc cgtgacattg
agtctctgga caagaagatc cgcccgctgg aggagttaga 2880ggccctgctg
agcagcggca acaccaagca gctgaagaac aaggaagttg cagcgctggt
2940gatccacggt aagctgccac tgtatgcgct ggaaaagaaa ctgggcgata
cgacgcgtgc 3000ggtcgcggtg cgtcgcaaag ccttaagcat cttagcggag
gccccggtgt tagccagcga 3060ccgcctgccg tacaagaact acgactacga
ccgcgtgttt ggcgcgtgct gcgagaatgt 3120cattggctac atgccgttac
cggttggtgt gatcggcccg ctggtcattg atggcacgag 3180ctatcacatt
ccaatggcga ccacggaagg ttgcttagtc gccagcgcca tgcgtggctg
3240taaggcgatt aacgccggcg gtggcgcgac gaccgtgtta accaaggatg
gtatgacgcg 3300cggtccggtc gtccgcttcc caacgctgaa gcgcagcggc
gcgtgtaaga tttggctgga 3360ttctgaggag ggccaaaacg cgatcaagaa
agccttcaac tctacgagcc gtttcgcgcg 3420tttacagcat atccagacct
gcctggccgg cgacctgctg ttcatgcgct tccgcaccac 3480cacgggcgat
gcgatgggca tgaacatgat cagcaagggc gtcgaatata gcctgaaaca
3540aatggtggaa gaatatggct gggaggacat ggaggttgtc tctgtgagcg
gcaactattg 3600caccgacaag aagccggcag ccattaactg gattgagggt
cgcggcaaaa gcgtcgtggc 3660agaagcgacc atcccaggcg acgtggtccg
taaggttctg aagagcgacg tcagcgccct 3720ggttgagtta aatatcgcga
aaaacctggt cggcagcgcg atggcgggca gcgtgggtgg 3780ctttaacgca
catgcagcga atctggttac ggcggttttc ttagccttag gtcaggaccc
3840agcccaaaat gtcgagagca gcaactgcat taccttaatg aaagaggttg
acggtgacct 3900gcgcatcagc gtttctatgc cgtctatcga ggtcggcacg
atcggcggcg gcaccgtttt 3960agaaccgcaa ggtgcgatgc tggatctgct
gggcgtgcgc ggcccacatg caacggcccc 4020aggcaccaat gcccgccaac
tggcccgtat cgtggcctgc gcggttctgg cgggtgagct 4080gagcctgtgc
gccgcattag ccgcgggcca tttagttcaa tctcacatga cccacaaccg
4140caagccggca gaaccaacca agccaaataa cctggacgca accgacatta
accgtctgaa 4200ggatggcagc gtcacgtgca ttaaaagctg agcatgctac taagctt
4247249DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2gctactagta ggaggaaaac atcatgcaaa gtttagataa
gaatttccg 49339DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 3gcttctagac tattgttgtc taatttcttg
taaaatgcg 39455DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 4gaactgaaga tctaggagga aagcaaaatg
acaataggta tcgacaaaat aaact 55552DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 5ttgcatgatg ttttcctcct
actagttact ctggtctgtg atattcgcga ac 52642DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6gctaggccat cctggccatg aagaactgtg tgattgtttc tg 42733DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7gcttgcgatc gccggcggat ttgtcctact cag 33835DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8ccacctcgag atgtcattac cgttcttaac ttctg 35941DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9tggtggagct cttatttaag ctgggtaaat gcagataatc g 411039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10ttcttgagct cttattcctt tggtagacca gtctttgcg 391151DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11tatggatcct aaggaggata tttagatgaa aacagtagtt attattgatg c
511240DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12agctaagctt ttattgtttt cttaaatcat ttaaaatagc
401351DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13tatagatctt aaggaggata tttagatgac aattgggatt
gataaaatta g 511432DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 14tttggatcct tagtttcgat aagagcgaac gg
321545DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15acactcgagg aggaataaat gagttttgat attgccaaat
acccg 451637DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 16tgatggtacc ttatgccagc caggccttga
ttttggc 371745DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 17actaggtacc aggaggaata aatgaagcaa
ctcaccattc tgggc 451837DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18aattgatggg ccctcagctt
gcgagacgca tcacctc 371945DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 19cataaagggc ccaggaggaa
taaatggcaa ccactcattt ggatg 452040DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 20tattgttcat atgttatgta
ttctcctgat ggatggttcg 402145DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21aactaacaca tatgaggagg
aataaatgcg gacacagtgg ccctc 452238DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 22tgttagttac gcgtttaaag
catggctctg tgcaatgg 382345DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 23acgggatcca ggaggaataa
atgcgaattg gacacggttt tgacg 452441DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 24tttagttggg ccctcatttt
gttgccttaa tgagtagcgc c 412549DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 25tactaagggc ccaggaggaa
ataatgcata accaggctcc aattcaacg 492640DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26tccgggtacc ttatttttca acctgctgaa cgtcaattcg 402743DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27aacaggtacc aggaggaaat aatgcagatc ctgttggcca acc
432845DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28tggatgaagt cgacttaatc gacttcacga atatcgacac
gcagc 452950DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 29catcaagtcg acaggaggaa ataatgcaaa
cggaacacgt cattttattg 503040DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 30taatgcaagc ttatttaagc
tgggtaaatg cagataatcg 403144DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 31cagtaaagct taggaggaaa
taatggactt tccgcagcaa ctcg 443241DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 32tagttccatg gttatttatt
acgctggatg atgtagtccg c 413335DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 33acatagacgt cgggaaagcg
aggatctagg taggg 353437DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 34ttcccgctcg aggtggcgga
ccatataggc agatcag 373523DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 35gacgtcgata tctggcgaaa atg
233622DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 36tactagtgct tggattctca cc 223732DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37ccatggacac tctgccgatc tcttccgtaa gc 323828DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38gagctctcat acgaccatag ggtgtacg 283925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39ccatggacct ggcagtagaa attgc 254027DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
40gagctcttac atcggtaccg gctccag 27413978DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
41atgaagaact gtgtgattgt ttctgcggtc cgcacggcga tcggcagctt taacggctct
60ttagcgagca cctctgcaat cgatctgggt gcgacggtca ttaaggccgc cattgaacgc
120gccaaaatcg acagccagca cgttgatgag gtgatcatgg gcaatgtgtt
acaagccggc 180ctgggtcaaa acccagcgcg tcaagcactg ttaaaatctg
gtctggccga gaccgtgtgt 240ggcttcaccg tcaataaggt ttgcggctct
ggcctgaaga gcgtggccct ggcagcacaa 300gcgattcaag ccggtcaggc
acaaagcatc gttgcgggtg gcatggagaa catgtctctg 360gcgccgtact
tattagatgc caaagcccgc agcggttatc gcctgggcga tggtcaggtg
420tacgacgtca tcttacgcga tggcttaatg tgcgcgaccc acggttacca
catgggtatt 480acggccgaaa acgtggcgaa agaatacggc attacgcgcg
agatgcagga tgaattagca 540ctgcactctc agcgcaaagc agcagccgcg
atcgagtctg gtgcgtttac ggcggaaatc 600gtgccagtta acgtggtcac
gcgcaagaag acgttcgttt tcagccagga cgagttcccg 660aaggcaaaca
gcaccgcgga ggccttaggt gccttacgcc cagcctttga caaagcgggc
720acggtcaccg ccggtaatgc gagcggcatc aatgatggtg cagcggcact
ggtcatcatg 780gaagagagcg ccgcattagc agcgggtctg accccattag
cgcgcattaa atcttatgcc 840agcggcggcg tcccaccagc cctgatgggc
atgggtccgg tcccagccac gcaaaaagcc 900ctgcaattag cgggcctgca
actggccgac attgatctga tcgaggcgaa cgaggcgttt 960gcagcgcagt
tcctggcggt gggtaagaat ctgggcttcg acagcgagaa agtcaatgtg
1020aacggtggcg cgattgcgtt aggccatccg attggtgcaa gcggcgcacg
catcttagtg 1080acgttactgc acgccatgca ggcacgcgac aagaccttag
gcctggcgac cttatgtatt 1140ggtggcggtc aaggtatcgc catggtgatc
gaacgcctga actgaagatc taggaggaaa 1200gcaaaatgaa actgagcacc
aagctgtgct ggtgtggcat caagggtcgc ctgcgcccac 1260aaaagcagca
acagctgcac aacacgaacc tgcaaatgac cgagctgaaa aagcagaaga
1320cggccgagca aaagacccgc ccgcagaacg ttggcatcaa gggcatccag
atttatatcc 1380cgacgcagtg tgtcaaccaa tctgagctgg agaaattcga
tggcgtcagc cagggtaagt 1440acaccatcgg cctgggccag accaacatga
gcttcgtgaa cgaccgtgag gacatctatt 1500ctatgagcct gacggtgctg
tctaagctga tcaagagcta caacatcgac acgaataaga 1560tcggtcgtct
ggaggtgggt acggagacgc tgattgacaa gagcaaaagc gtgaagtctg
1620tcttaatgca gctgttcggc gagaacacgg atgtcgaggg tatcgacacc
ctgaacgcgt 1680gttacggcgg caccaacgca ctgttcaata gcctgaactg
gattgagagc aacgcctggg 1740atggccgcga tgcgatcgtc gtgtgcggcg
atatcgccat ctatgacaag ggtgcggcac 1800gtccgaccgg cggtgcaggc
accgttgcga tgtggattgg cccggacgca ccaattgtct 1860tcgattctgt
ccgcgcgtct tacatggagc acgcctacga cttttacaag ccggacttca
1920cgagcgaata cccgtacgtg gacggccact tctctctgac ctgctatgtg
aaggcgctgg 1980accaggttta taagtcttat agcaaaaagg cgatttctaa
gggcctggtc agcgacccgg 2040caggcagcga cgccctgaac gtgctgaagt
atttcgacta caacgtgttc catgtcccga 2100cctgcaaatt agtgaccaaa
tcttatggcc gcctgttata taatgatttc cgtgccaacc 2160cgcagctgtt
cccggaggtt gacgccgagc tggcgacgcg tgattacgac gagagcctga
2220ccgacaagaa catcgagaag accttcgtca acgtcgcgaa gccgttccac
aaagagcgtg 2280tggcccaaag cctgatcgtc ccgaccaaca cgggcaacat
gtataccgcg tctgtctacg 2340cggcattcgc gagcctgctg aattacgtcg
gttctgacga cctgcagggc aagcgcgttg 2400gcctgttcag ctacggtagc
ggcttagcgg ccagcctgta tagctgcaaa attgtcggcg 2460acgtccagca
catcatcaag gagctggaca tcaccaacaa gctggcgaag cgcatcaccg
2520agacgccgaa agattacgag gcagcgatcg agttacgcga gaatgcgcat
ctgaagaaga 2580acttcaagcc gcaaggtagc atcgagcacc tgcagagcgg
cgtctactac ctgacgaaca 2640ttgacgacaa gttccgccgt tcttatgacg
tcaaaaagta actagtagga ggaaaacatc 2700atgcaaagtt tagataagaa
tttccgacat ttatctcgtc aacaaaagtt acaacaattg 2760gtagataagc
aatggttatc agaagatcaa ttcgacattt tattgaatca tccattaatt
2820gatgaggaag tagcaaatag tttaattgaa aatgtcatcg cgcaaggtgc
attacccgtt 2880ggattattac cgaatatcat tgtggacgat aaggcatatg
ttgtacctat gatggtggaa 2940gagccttcag ttgtcgctgc agctagttat
ggtgcaaagc tagtgaatca gactggcgga 3000tttaaaacgg tatcttctga
acgtattatg ataggtcaaa tcgtctttga tggcgttgac 3060gatactgaaa
aattatcagc agacattaaa gctttagaaa agcaaattca taaaattgcg
3120gatgaggcat atccttctat taaagcgcgt ggtggtggtt accaacgtat
agctattgat 3180acatttcctg agcaacagtt actatcttta aaagtatttg
ttgatacgaa agatgctatg 3240ggcgctaata tgcttaatac gattttagag
gccataactg catttttaaa aaatgaatct 3300ccacaaagcg acattttaat
gagtatttta tccaatcatg caacagcgtc cgttgttaaa 3360gttcaaggcg
aaattgacgt taaagattta gcaaggggcg agagaactgg agaagaggtt
3420gccaaacgaa tggaacgtgc ttctgtattg gcacaagttg atattcatcg
tgctgcaaca 3480cataataaag gtgttatgaa tggcatacat gccgttgttt
tagcaacagg aaatgatacg 3540cgtggtgcag aagcaagtgc gcatgcatac
gcgagtcgtg acggacagta tcgtggtatt 3600gcaacatgga gatacgatca
aaaacgtcaa cgtttaattg gtacaataga agtgcctatg 3660acattggcaa
tcgttggcgg tggtacaaaa gtattaccaa ttgctaaagc ttctttagaa
3720ttgctaaatg tagattcagc acaagaatta ggtcatgtag ttgctgccgt
tggtttagca 3780cagaactttg cagcatgtcg cgcgctcgtt tccgaaggta
tccagcaagg ccatatgagc 3840ttgcaatata aatctttagc tattgttgta
ggtgcaaaag gtgatgaaat tgcgcaagta 3900gctgaagcat tgaagcaaga
accccgtgcg aatacacaag tagctgaacg cattttacaa 3960gaaattagac aacaatag
3978423669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 42atgaagaact gtgtgattgt ttctgcggtc
cgcacggcga tcggcagctt taacggctct 60ttagcgagca cctctgcaat cgatctgggt
gcgacggtca ttaaggccgc cattgaacgc 120gccaaaatcg acagccagca
cgttgatgag gtgatcatgg gcaatgtgtt acaagccggc 180ctgggtcaaa
acccagcgcg tcaagcactg ttaaaatctg gtctggccga gaccgtgtgt
240ggcttcaccg tcaataaggt ttgcggctct ggcctgaaga gcgtggccct
ggcagcacaa 300gcgattcaag ccggtcaggc acaaagcatc gttgcgggtg
gcatggagaa catgtctctg 360gcgccgtact tattagatgc caaagcccgc
agcggttatc gcctgggcga tggtcaggtg 420tacgacgtca tcttacgcga
tggcttaatg tgcgcgaccc acggttacca catgggtatt 480acggccgaaa
acgtggcgaa agaatacggc attacgcgcg agatgcagga tgaattagca
540ctgcactctc agcgcaaagc agcagccgcg atcgagtctg gtgcgtttac
ggcggaaatc 600gtgccagtta acgtggtcac gcgcaagaag acgttcgttt
tcagccagga cgagttcccg 660aaggcaaaca gcaccgcgga ggccttaggt
gccttacgcc cagcctttga caaagcgggc 720acggtcaccg ccggtaatgc
gagcggcatc aatgatggtg cagcggcact ggtcatcatg 780gaagagagcg
ccgcattagc agcgggtctg accccattag cgcgcattaa atcttatgcc
840agcggcggcg tcccaccagc cctgatgggc atgggtccgg tcccagccac
gcaaaaagcc 900ctgcaattag cgggcctgca actggccgac attgatctga
tcgaggcgaa cgaggcgttt 960gcagcgcagt tcctggcggt gggtaagaat
ctgggcttcg acagcgagaa agtcaatgtg 1020aacggtggcg cgattgcgtt
aggccatccg attggtgcaa gcggcgcacg catcttagtg 1080acgttactgc
acgccatgca ggcacgcgac aagaccttag gcctggcgac cttatgtatt
1140ggtggcggtc aaggtatcgc catggtgatc gaacgcctga actgaagatc
taggaggaaa 1200gcaaaatgac aataggtatc gacaaaataa acttttacgt
tccaaagtac tatgtagaca 1260tggctaaatt agcagaagca cgccaagtag
acccaaacaa atttttaatt ggaattggtc
1320aaactgaaat ggctgttagt cctgtaaacc aagacatcgt ttcaatgggc
gctaacgctg 1380ctaaggacat tataacagac gaagataaaa agaaaattgg
tatggtaatt gtggcaactg 1440aatcagcagt tgatgctgct aaagcagccg
ctgttcaaat tcacaactta ttaggtattc 1500aaccttttgc acgttgcttt
gaaatgaaag aagcttgtta tgctgcaaca ccagcaattc 1560aattagctaa
agattattta gcaactagac cgaatgaaaa agtattagtt attgctacag
1620atacagcacg ttatggattg aattcaggcg gcgagccaac acaaggtgct
ggcgcagttg 1680cgatggttat tgcacataat ccaagcattt tggcattaaa
tgaagatgct gttgcttaca 1740ctgaagacgt ttatgatttc tggcgtccaa
ctggacataa atatccatta gttgatggtg 1800cattatctaa agatgcttat
atccgctcat tccaacaaag ctggaatgaa tacgcaaaac 1860gtcaaggtaa
gtcgctagct gacttcgcat ctctatgctt ccatgttcca tttacaaaaa
1920tgggtaaaaa ggcattagag tcaatcattg ataacgctga tgaaacaact
caagagcgtt 1980tacgttcagg atatgaagat gctgtagatt ataaccgtta
tgtcggtaat atttatactg 2040gatcattata tttaagccta atatcattac
ttgaaaatcg tgatttacaa gctggtgaaa 2100caatcggttt attcagttat
ggctcaggtt cagttggtga attttatagt gcgacattag 2160ttgaaggcta
caaagatcat ttagatcaag ctgcacataa agcattatta aataaccgta
2220ctgaagtatc tgttgatgca tatgaaacat tcttcaaacg ttttgatgac
gttgaatttg 2280acgaagaaca agatgctgtt catgaagatc gtcatatttt
ctacttatca aatattgaaa 2340ataacgttcg cgaatatcac agaccagagt
aactagtagg aggaaaacat catgcaaagt 2400ttagataaga atttccgaca
tttatctcgt caacaaaagt tacaacaatt ggtagataag 2460caatggttat
cagaagatca attcgacatt ttattgaatc atccattaat tgatgaggaa
2520gtagcaaata gtttaattga aaatgtcatc gcgcaaggtg cattacccgt
tggattatta 2580ccgaatatca ttgtggacga taaggcatat gttgtaccta
tgatggtgga agagccttca 2640gttgtcgctg cagctagtta tggtgcaaag
ctagtgaatc agactggcgg atttaaaacg 2700gtatcttctg aacgtattat
gataggtcaa atcgtctttg atggcgttga cgatactgaa 2760aaattatcag
cagacattaa agctttagaa aagcaaattc ataaaattgc ggatgaggca
2820tatccttcta ttaaagcgcg tggtggtggt taccaacgta tagctattga
tacatttcct 2880gagcaacagt tactatcttt aaaagtattt gttgatacga
aagatgctat gggcgctaat 2940atgcttaata cgattttaga ggccataact
gcatttttaa aaaatgaatc tccacaaagc 3000gacattttaa tgagtatttt
atccaatcat gcaacagcgt ccgttgttaa agttcaaggc 3060gaaattgacg
ttaaagattt agcaaggggc gagagaactg gagaagaggt tgccaaacga
3120atggaacgtg cttctgtatt ggcacaagtt gatattcatc gtgctgcaac
acataataaa 3180ggtgttatga atggcataca tgccgttgtt ttagcaacag
gaaatgatac gcgtggtgca 3240gaagcaagtg cgcatgcata cgcgagtcgt
gacggacagt atcgtggtat tgcaacatgg 3300agatacgatc aaaaacgtca
acgtttaatt ggtacaatag aagtgcctat gacattggca 3360atcgttggcg
gtggtacaaa agtattacca attgctaaag cttctttaga attgctaaat
3420gtagattcag cacaagaatt aggtcatgta gttgctgccg ttggtttagc
acagaacttt 3480gcagcatgtc gcgcgctcgt ttccgaaggt atccagcaag
gccatatgag cttgcaatat 3540aaatctttag ctattgttgt aggtgcaaaa
ggtgatgaaa ttgcgcaagt agctgaagca 3600ttgaagcaag aaccccgtgc
gaatacacaa gtagctgaac gcattttaca agaaattaga 3660caacaatag
3669432235DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 43caataccgac ttaccatcct atttgctttg
ccctttttct tttccactgc atggcggcgt 60tagtatcgaa tggatggcgg cgttagtatc
gaatcgacag cagtatagcg accagcattc 120acatacgatt gacgcatgat
attactttct gcgcacttaa cttcgcatct gggcagatga 180tgtcgaggcg
aaaaaaaata taaatcacgc taacatttga ttaaaataga acaactacaa
240tataaaaaaa ctatacaaat gacaagttct tgaaaacaag aatcttttta
ttgtcagtac 300tgattagaaa aactcatcga gcatcaaatg aaactgcaat
ttattcatat caggattatc 360aataccatat ttttgaaaaa gccgtttctg
taatgaagga gaaaactcac cgaggcagtt 420ccataggatg gcaagatcct
ggtatcggtc tgcgattccg actcgtccaa catcaataca 480acctattaat
ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
540gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
gttcaacagg 600ccagccatta cgctcgtcat caaaatcact cgcatcaacc
aaaccgttat tcattcgtga 660ttgcgcctga gcgagacgaa atacgcgatc
gctgttaaaa ggacaattac aaacaggaat 720cgaatgcaac cggcgcagga
acactgccag cgcatcaaca atattttcac ctgaatcagg 780atattcttct
aatacctgga atgctgtttt gccggggatc gcagtggtga gtaaccatgc
840atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
ccgtcagcca 900gtttagtctg accatctcat ctgtaacatc attggcaacg
ctacctttgc catgtttcag 960aaacaactct ggcgcatcgg gcttcccata
caatcgatag attgtcgcac ctgattgccc 1020gacattatcg cgagcccatt
tatacccata taaatcagca tccatgttgg aatttaatcg 1080cggcctcgaa
acgtgagtct tttccttacc catggttgtt tatgttcgga tgtgatgtga
1140gaactgtatc ctagcaagat tttaaaagga agtatatgaa agaagaacct
cagtggcaaa 1200tcctaacctt ttatatttct ctacaggggc gcggcgtggg
gacaattcaa cgcgtctgtg 1260aggggagcgt ttccctgctc gcaggtctgc
agcgaggagc cgtaattttt gcttcgcgcc 1320gtgcggccat caaaatgtat
ggatgcaaat gattatacat ggggatgtat gggctaaatg 1380tacgggcgac
agtcacatca tgcccctgag ctgcgcacgt caagactgtc aaggagggta
1440ttctgggcct ccatgtcgct ggccgggtga cccggcgggg acgaggcaag
ctaaacagat 1500ctgatcttga aactgagtaa gatgctcaga atacccgtca
agataagagt ataatgtaga 1560gtaatatacc aagtattcag catattctcc
tcttcttttg tataaatcac ggaagggatg 1620atttataaga aaaatgaata
ctattacact tcatttacca ccctctgatc tagattttcc 1680aacgatatgt
acgtagtggt ataaggtgag ggggtccaca gatataacat cgtttaattt
1740agtactaaca gagacttttg tcacaactac atataagtgt acaaatatag
tacagatatg 1800acacacttgt agcgccaacg cgcatcctac ggattgctga
cagaaaaaaa ggtcacgtga 1860ccagaaaagt cacgtgtaat tttgtaactc
accgcattct agcggtccct gtcgtgcaca 1920ctgcactcaa caccataaac
cttagcaacc tccaaaggaa atcaccgtat aacaaagcca 1980cagttttaca
acttagtctc ttatgaagtt acttaccaat gagaaataga ggctctttct
2040cgagaaatat gaatatggat atatatatat atatatatat atatatatat
atatgtaaac 2100ttggttcttt tttagcttgt gatctctagc ttgggtctct
ctctgtcgta acagttgtga 2160tatcggctgc cttcatctcg accggatgca
atgccaattg taatagcttt cccatgttaa 2220ttatacttta ttctt
22354466DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44gagtgaacct gctgcctggc gtgctctgac
tcagtacatt tcatagtgga tggcggcgtt 60agtatc 664565DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 45cgtgtatacg ttttccgctt ctgctcttcg tcttttctct
tcttccgata tcacaactgt 60tacga 654670DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 46ggtaagacgg ttgggtttta tcttttgcag ttggtactat
taagaacaat cacaggaaac 60agctatgacc 704770DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 47ttgcgttttg tactttggtt cgctcaattt tgcaggtaga
taatcgaaaa gttgtaaaac 60gacggccagt 70485487DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
48gtttaaactt gctaaattcg agtgaaacac aggaagacca gaaaatcctc atttcatcca
60tattaacaat aatttcaaat gtttatttgc attatttgaa actagggaag acaagcaacg
120aaacgttttg aaaattttga gtattttcaa taaatttgta gaggactcag
atattgaaaa 180aaagctacag caattaatac ttgataagaa gagtattgag
aagggcaacg gttcatcatc 240tcatggatct gcacatgaac aaacaccaga
gtcaaacgac gttgaaattg aggctactgc 300gccaattgat gacaatacag
acgatgataa caaaccgaag ttatctgatg tagaaaagga 360ttaaagatgc
taagagatag tgatgatatt tcataaataa tgtaattcta tatatgttaa
420ttaccttttt tgcgaggcat atttatggtg aaggataagt tttgaccatc
aaagaaggtt 480aatgtggctg tggtttcagg gtccataccc gggagttatg
acaattacaa caacagaatt 540ctttctatat atgcacgaac ttgtaatatg
gaagaaatta tgacgtacaa actataaagt 600aaatatttta cgtaacacat
ggtgctgttg tgcttctttt tcaagagaat accaatgacg 660tatgactaag
tttaggattt aatgcaggtg acggacccat ctttcaaacg atttatatca
720gtggcgtcca aattgttagg ttttgttggt tcagcaggtt tcctgttgtg
ggtcatatga 780ctttgaacca aatggccggc tgctagggca gcacataagg
ataattcacc tgccaagacg 840gcacaggcaa ctattcttgc taattgacgt
gcgttggtac caggagcggt agcatgtggg 900cctcttacac ctaataagtc
caacatggca ccttgtggtt ctagaacagt accaccaccg 960atggtaccta
cttcgatgga tggcatggat acggaaattc tcaaatcacc gtccacttct
1020ttcatcaatg ttatacagtt ggaactttcg acattttgtg caggatcttg
tcctaatgcc 1080aagaaaacag ctgtcactaa attagctgca tgtgcgttaa
atccaccaac agacccagcc 1140attgcagatc caaccaaatt cttagcaatg
ttcaactcaa ccaatgcgga aacatcactt 1200tttaacactt ttctgacaac
atcaccagga atagtagctt ctgcgacgac actcttacca 1260cgaccttcga
tccagttgat ggcagctggt tttttgtcgg tacagtagtt accagaaacg
1320gagacaacct ccatatcttc ccagccatac tcttctacca tttgctttaa
tgagtattcg 1380acacccttag aaatcatatt catacccatt gcgtcaccag
tagttgttct aaatctcatg 1440aagagtaaat ctcctgctag acaagtttga
atatgttgca gacgtgcaaa tcttgatgta 1500gagttaaaag cttttttaat
tgcgttttgt ccctcttctg agtctaacca tatcttacag 1560gcaccagatc
ttttcaaagt tgggaaacgg actactgggc ctcttgtcat accatcctta
1620gttaaaacag ttgttgcacc accgccagca ttgattgcct tacagccacg
catggcagaa 1680gctaccaaac aaccctctgt agttgccatt ggtatatgat
aagatgtacc atcgataacc 1740aaggggccta taacaccaac gggcaaaggc
atgtaaccta taacattttc acaacaagcg 1800ccaaatacgc ggtcgtagtc
ataattttta tatggtaaac gatcagatgc taatacagga 1860gcttctgcca
aaattgaaag agccttccta cgtaccgcaa ccgctctcgt agtatcacct
1920aattttttct ccaaagcgta caaaggtaac ttaccgtgaa taaccaaggc
agcgacctct 1980ttgttcttca attgttttgt atttccacta cttaataatg
cttctaattc ttctaaagga 2040cgtattttct tatccaagct ttcaatatcg
cgggaatcat cttcctcact agatgatgaa 2100ggtcctgatg agctcgattg
cgcagatgat aaacttttga ctttcgatcc agaaatgact 2160gttttattgg
ttaaaactgg tgtagaagcc ttttgtacag gagcagtaaa agacttcttg
2220gtgacttcag tcttcaccaa ttggtctgca gccattatag ttttttctcc
ttgacgttaa 2280agtatagagg tatattaaca attttttgtt gatactttta
tgacatttga ataagaagta 2340atacaaaccg aaaatgttga aagtattagt
taaagtggtt atgcagcttt tgcatttata 2400tatctgttaa tagatcaaaa
atcatcgctt cgctgattaa ttaccccaga aataaggcta 2460aaaaactaat
cgcattatta tcctatggtt gttaatttga ttcgttgatt tgaaggtttg
2520tggggccagg ttactgccaa tttttcctct tcataaccat aaaagctagt
attgtagaat 2580ctttattgtt cggagcagtg cggcgcgagg cacatctgcg
tttcaggaac gcgaccggtg 2640aagaccagga cgcacggagg agagtcttcc
gtcggagggc tgtcgcccgc tcggcggctt 2700ctaatccgta cttcaatata
gcaatgagca gttaagcgta ttactgaaag ttccaaagag 2760aaggtttttt
taggctaaga taatggggct ctttacattt ccacaacata taagtaagat
2820tagatatgga tatgtatatg gtggtattgc catgtaatat gattattaaa
cttctttgcg 2880tccatccaaa aaaaaagtaa gaatttttga aaattcaata
taaatgaaac tctcaactaa 2940actttgttgg tgtggtatta aaggaagact
taggccgcaa aagcaacaac aattacacaa 3000tacaaacttg caaatgactg
aactaaaaaa acaaaagacc gctgaacaaa aaaccagacc 3060tcaaaatgtc
ggtattaaag gtatccaaat ttacatccca actcaatgtg tcaaccaatc
3120tgagctagag aaatttgatg gcgtttctca aggtaaatac acaattggtc
tgggccaaac 3180caacatgtct tttgtcaatg acagagaaga tatctactcg
atgtccctaa ctgttttgtc 3240taagttgatc aagagttaca acatcgacac
caacaaaatt ggtagattag aagtcggtac 3300tgaaactctg attgacaagt
ccaagtctgt caagtctgtc ttgatgcaat tgtttggtga 3360aaacactgac
gtcgaaggta ttgacacgct taatgcctgt tacggtggta ccaacgcgtt
3420gttcaactct ttgaactgga ttgaatctaa cgcatgggat ggtagagacg
ccattgtagt 3480ttgcggtgat attgccatct acgataaggg tgccgcaaga
ccaaccggtg gtgccggtac 3540tgttgctatg tggatcggtc ctgatgctcc
aattgtattt gactctgtaa gagcttctta 3600catggaacac gcctacgatt
tttacaagcc agatttcacc agcgaatatc cttacgtcga 3660tggtcatttt
tcattaactt gttacgtcaa ggctcttgat caagtttaca agagttattc
3720caagaaggct atttctaaag ggttggttag cgatcccgct ggttcggatg
ctttgaacgt 3780tttgaaatat ttcgactaca acgttttcca tgttccaacc
tgtaaattgg tcacaaaatc 3840atacggtaga ttactatata acgatttcag
agccaatcct caattgttcc cagaagttga 3900cgccgaatta gctactcgcg
attatgacga atctttaacc gataagaaca ttgaaaaaac 3960ttttgttaat
gttgctaagc cattccacaa agagagagtt gcccaatctt tgattgttcc
4020aacaaacaca ggtaacatgt acaccgcatc tgtttatgcc gcctttgcat
ctctattaaa 4080ctatgttgga tctgacgact tacaaggcaa gcgtgttggt
ttattttctt acggttccgg 4140tttagctgca tctctatatt cttgcaaaat
tgttggtgac gtccaacata ttatcaagga 4200attagatatt actaacaaat
tagccaagag aatcaccgaa actccaaagg attacgaagc 4260tgccatcgaa
ttgagagaaa atgcccattt gaagaagaac ttcaaacctc aaggttccat
4320tgagcatttg caaagtggtg tttactactt gaccaacatc gatgacaaat
ttagaagatc 4380ttacgatgtt aaaaaataat cttcccccat cgattgcatc
ttgctgaacc cccttcataa 4440atgctttatt tttttggcag cctgcttttt
ttagctctca tttaatagag tagtttttta 4500atctatatac taggaaaact
ctttatttaa taacaatgat atatatatac ccgggaagct 4560tttcaattca
tctttttttt ttttgttctt ttttttgatt ccggtttctt tgaaattttt
4620ttgattcggt aatctccgag cagaaggaag aacgaaggaa ggagcacaga
cttagattgg 4680tatatatacg catatgtggt gttgaagaaa catgaaattg
cccagtattc ttaacccaac 4740tgcacagaac aaaaacctgc aggaaacgaa
gataaatcat gtcgaaagct acatataagg 4800aacgtgctgc tactcatcct
agtcctgttg ctgccaagct atttaatatc atgcacgaaa 4860agcaaacaaa
cttgtgtgct tcattggatg ttcgtaccac caaggaatta ctggagttag
4920ttgaagcatt aggtcccaaa atttgtttac taaaaacaca tgtggatatc
ttgactgatt 4980tttccatgga gggcacagtt aagccgctaa aggcattatc
cgccaagtac aattttttac 5040tcttcgaaga cagaaaattt gctgacattg
gtaatacagt caaattgcag tactctgcgg 5100gtgtatacag aatagcagaa
tgggcagaca ttacgaatgc acacggtgtg gtgggcccag 5160gtattgttag
cggtttgaag caggcggcgg aagaagtaac aaaggaacct agaggccttt
5220tgatgttagc agaattgtca tgcaagggct ccctagctac tggagaatat
actaagggta 5280ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg
ctttattgct caaagagaca 5340tgggtggaag agatgaaggt tacgattggt
tgattatgac acccggtgtg ggtttagatg 5400acaagggaga cgcattgggt
caacagtata gaaccgtgga tgatgtggtc tctacaggat 5460ctgacattat
tattgttggg tttaaac 5487495860DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 49gtttaaactt
gctaaattcg agtgaaacac aggaagacca gaaaatcctc atttcatcca 60tattaacaat
aatttcaaat gtttatttgc attatttgaa actagggaag acaagcaacg
120aaacgttttt gaaaattttg agtattttca ataaatttgt agaggactca
gatattgaaa 180aaaagctaca gcaattaata cttgataaga agagtattga
gaagggcaac ggttcatcat 240ctcatggatc tgcacatgaa caaacaccag
agtcaaacga cgttgaaatt gaggctactg 300cgccaattga tgacaataca
gacgatgata acaaaccgaa gttatctgat gtagaaaagg 360attaaagatg
ctaagagata gtgatgatat ttcataaata atgtaattct atatatgtta
420attacctttt ttgcgaggca tatttatggt gaaggataag ttttgaccat
caaagaaggt 480taatgtggct gtggtttcag ggtccatacc cgggtatata
tatatcattg ttattaaata 540aagagttttc ctagtatata gattaaaaaa
ctactctatt aaatgagagc taaaaaaagc 600aggctgccaa aaaaataaag
catttatgaa gggggttcag caagatgcaa tcgatggggg 660aagattattt
tttaacatcg taagatcttc taaatttgtc atcgatgttg gtcaagtagt
720aaacaccact ttgcaaatgc tcaatggaac cttgaggttt gaagttcttc
ttcaaatggg 780cattttctct caattcgatg gcagcttcgt aatcctttgg
agtttcggtg attctcttgg 840ctaatttgtt agtaatatct aattccttga
taatatgttg gacgtcacca acaattttgc 900aagaatatag agatgcagct
aaaccggaac cgtaagaaaa taaaccaaca cgcttgcctt 960gtaagtcgtc
agatccaaca tagtttaata gagatgcaaa ggcggcataa acagatgcgg
1020tgtacatgtt acctgtgttt gttggaacaa tcaaagattg ggcaactctc
tctttgtgga 1080atggcttagc aacattaaca aaagtttttt caatgttctt
atcggttaaa gattcgtcat 1140aatcgcgagt agctaattcg gcgtcaactt
ctgggaacaa ttgaggattg gctctgaaat 1200cgttatatag taatctaccg
tatgattttg tgaccaattt acaggttgga acatggaaaa 1260cgttgtagtc
gaaatatttc aaaacgttca aagcatccga accagcggga tcgctaacca
1320accctttaga aatagccttc ttggaataac tcttgtaaac ttgatcaaga
gccttgacgt 1380aacaagttaa tgaaaaatga ccatcgacgt aaggatattc
gctggtgaaa tctggcttgt 1440aaaaatcgta ggcgtgttcc atgtaagaag
ctcttacaga gtcaaataca attggagcat 1500caggaccgat ccacatagca
acagtaccgg caccaccggt tggtcttgcg gcacccttat 1560cgtagatggc
aatatcaccg caaactacaa tggcgtctct accatcccat gcgttagatt
1620caatccagtt caaagagttg aacaacgcgt tggtaccacc gtaacaggca
ttaagcgtgt 1680caataccttc gacgtcagtg ttttcaccaa acaattgcat
caagacagac ttgacagact 1740tggacttgtc aatcagagtt tcagtaccga
cttctaatct accaattttg ttggtgtcga 1800tgttgtaact cttgatcaac
ttagacaaaa cagttaggga catcgagtag atatcttctc 1860tgtcattgac
aaaagacatg ttggtttggc ccagaccaat tgtgtattta ccttgagaaa
1920cgccatcaaa tttctctagc tcagattggt tgacacattg agttgggatg
taaatttgga 1980tacctttaat accgacattt tgaggtctgg ttttttgttc
agcggtcttt tgttttttta 2040gttcagtcat ttgcaagttt gtattgtgta
attgttgttg cttttgcggc ctaagtcttc 2100ctttaatacc acaccaacaa
agtttagttg agagtttcat tttatgtgat gattgattga 2160ttgattgtac
agtttgtttt tcttaatatc tatttcgatg acttctatat gatattgcac
2220taacaagaag atattataat gcaattgata caagacaagg agttatttgc
ttctctttta 2280tatgattctg acaatccata ttgcgttggt agtctttttt
gctggaacgg ttcagcggaa 2340aagacgcatc gctctttttg cttctagaag
aaatgccagc aaaagaatct cttgacagtg 2400actgacagca aaaatgtctt
tttctaacta gtaacaaggc taagatatca gcctgaaata 2460aagggtggtg
aagtaataat taaatcatcc gtataaacct atacacatat atgaggaaaa
2520ataatacaaa agtgttttaa atacagatac atacatgaac atatgcacgt
atagcgccca 2580aatgtcggta atgggatcgg cttactaatt ataaaatgca
tcatagaaat cgttgaagtt 2640gacgcagcga ctcgagatcc ataggagcaa
ctcatgtctg aacttcaacg atttctatga 2700tgcattttat aattagtaag
ccgatcccat taccgacatt tgggcgctat acgtgcatat 2760gttcatgtat
gtatctgtat ttaaaacact tttgtattat ttttcctcat atatgtgtat
2820aggtttatac ggatgattta attattactt caccaccctt tatttcaggc
tgatatctta 2880gccttgttac tagttagaaa aagacatttt tgctgtcagt
cactgtcaag agattctttt 2940gctggcattt cttctagaag caaaaagagc
gatgcgtctt ttccgctgaa ccgttccagc 3000aaaaaagact accaacgcaa
tatggattgt cagaatcata taaaagagaa gcaaataact 3060ccttgtcttg
tatcaattgc attataatat cttcttgtta gtgcaatatc atatagaagt
3120catcgaaata gatattaaga aaaacaaact gtacaatcaa tcaatcaatc
atcacataaa 3180atggctgcag accaattggt gaagactgaa gtcaccaaga
agtcttttac tgctcctgta 3240caaaaggctt ctacaccagt tttaaccaat
aaaacagtca tttctggatc gaaagtcaaa 3300agtttatcat ctgcgcaatc
gagctcatca ggaccttcat catctagtga ggaagatgat 3360tcccgcgata
ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta
3420ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt
ggttattcac 3480ggtaagttac ctttgtacgc tttggagaaa aaattaggtg
atactacgag agcggttgcg 3540gtacgtagga aggctctttc aattttggca
gaagctcctg tattagcatc tgatcgttta 3600ccatataaaa attatgacta
cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 3660tacatgcctt
tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat
3720ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg
ctgtaaggca
3780atcaatgctg gcggtggtgc aacaactgtt ttaactaagg atggtatgac
aagaggccca 3840gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta
agatatggtt agactcagaa 3900gagggacaaa acgcaattaa aaaagctttt
aactctacat caagatttgc acgtctgcaa 3960catattcaaa cttgtctagc
aggagattta ctcttcatga gatttagaac aactactggt 4020gacgcaatgg
gtatgaatat gatttctaag ggtgtcgaat actcattaaa gcaaatggta
4080gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta
ctgtaccgac 4140aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta
agagtgtcgt cgcagaagct 4200actattcctg gtgatgttgt cagaaaagtg
ttaaaaagtg atgtttccgc attggttgag 4260ttgaacattg ctaagaattt
ggttggatct gcaatggctg ggtctgttgg tggatttaac 4320gcacatgcag
ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa
4380aatgtcgaaa gttccaactg tataacattg atgaaagaag tggacggtga
tttgagaatt 4440tccgtatcca tgccatccat cgaagtaggt accatcggtg
gtggtactgt tctagaacca 4500caaggtgcca tgttggactt attaggtgta
agaggcccac atgctaccgc tcctggtacc 4560aacgcacgtc aattagcaag
aatagttgcc tgtgccgtct tggcaggtga attatcctta 4620tgtgctgccc
tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct
4680gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt
gaaagatggg 4740tccgtcacct gcattaaatc ctaaacttag tcatacgtca
ttggtattct cttgaaaaag 4800aagcacaaca gcaccatgtg ttacgtaaaa
tatttacttt atagtttgta cgtcataatt 4860tcttccatat tacaagttcg
tgcatatata gaaagaattc tgttgttgta attgtcataa 4920ctcccgggaa
gcttttcaat tcatcttttt tttttttgtt cttttttttg attccggttt
4980ctttgaaatt tttttgattc ggtaatctcc gagcagaagg aagaacgaag
gaaggagcac 5040agacttagat tggtatatat acgcatatgt ggtgttgaag
aaacatgaaa ttgcccagta 5100ttcttaaccc aactgcacag aacaaaaacc
tgcaggaaac gaagataaat catgtcgaaa 5160gctacatata aggaacgtgc
tgctactcat cctagtcctg ttgctgccaa gctatttaat 5220atcatgcacg
aaaagcaaac aaacttgtgt gcttcattgg atgttcgtac caccaaggaa
5280ttactggagt tagttgaagc attaggtccc aaaatttgtt tactaaaaac
acatgtggat 5340atcttgactg atttttccat ggagggcaca gttaagccgc
taaaggcatt atccgccaag 5400tacaattttt tactcttcga agacagaaaa
tttgctgaca ttggtaatac agtcaaattg 5460cagtactctg cgggtgtata
cagaatagca gaatgggcag acattacgaa tgcacacggt 5520gtggtgggcc
caggtattgt tagcggtttg aagcaggcgg cggaagaagt aacaaaggaa
5580cctagaggcc ttttgatgtt agcagaattg tcatgcaagg gctccctagc
tactggagaa 5640tatactaagg gtactgttga cattgcgaag agcgacaaag
attttgttat cggctttatt 5700gctcaaagag acatgggtgg aagagatgaa
ggttacgatt ggttgattat gacacccggt 5760gtgggtttag atgacaaggg
agacgcattg ggtcaacagt atagaaccgt ggatgatgtg 5820gtctctacag
gatctgacat tattattgtt gggtttaaac 5860505050DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
50gtttaaacta ctattagctg aattgccact gctatcgttg ttagtggcgt tagtgcttgc
60attcaaagac atggagggcg ttattacgcc ggagctcctc gacagcagat ctgatgactg
120gtcaatatat ttttgcattg aggctctgtt tggaattata ttttgagatg
acccatctaa 180tgtactggta tcaccagatt tcatgtcgtt ttttaaagcg
gctgcttgag tcttagcaat 240agcgtcacca tctggtgaat cctttgaagg
aaccactgac gaaggtttgg acagtgacga 300agaggatctt tcctgctttg
aattagtcgc gctgggagca gatgacgagt tggtggagct 360gggggcagga
ttgctggccg tcgtgggtcc tgaatgggtc cttggctggt ccatctctat
420tctgaaaacg gaagaggagt agggaatatt actggctgaa aataagtctt
gaatgaacgt 480atacgcgtat atttctacca atctctcaac actgagtaat
ggtagttata agaaagagac 540cgagttaggg acagttagag gcggtggaga
tattccttat ggcatgtctg gcgatgataa 600aacttttcaa acggcagccc
cgatctaaaa gagctgacac ccgggagtta tgacaattac 660aacaacagaa
ttctttctat atatgcacga acttgtaata tggaagaaat tatgacgtac
720aaactataaa gtaaatattt tacgtaacac atggtgctgt tgtgcttctt
tttcaagaga 780ataccaatga cgtatgacta agtttaggat ttaatgcagg
tgacggaccc atctttcaaa 840cgatttatat cagtggcgtc caaattgtta
ggttttgttg gttcagcagg tttcctgttg 900tgggtcatat gactttgaac
caaatggccg gctgctaggg cagcacataa ggataattca 960cctgccaaga
cggcacaggc aactattctt gctaattgac gtgcgttggt accaggagcg
1020gtagcatgtg ggcctcttac acctaataag tccaacatgg caccttgtgg
ttctagaaca 1080gtaccaccac cgatggtacc tacttcgatg gatggcatgg
atacggaaat tctcaaatca 1140ccgtccactt ctttcatcaa tgttatacag
ttggaacttt cgacattttg tgcaggatct 1200tgtcctaatg ccaagaaaac
agctgtcact aaattagctg catgtgcgtt aaatccacca 1260acagacccag
ccattgcaga tccaaccaaa ttcttagcaa tgttcaactc aaccaatgcg
1320gaaacatcac tttttaacac ttttctgaca acatcaccag gaatagtagc
ttctgcgacg 1380acactcttac cacgaccttc gatccagttg atggcagctg
gttttttgtc ggtacagtag 1440ttaccagaaa cggagacaac ctccatatct
tcccagccat actcttctac catttgcttt 1500aatgagtatt cgacaccctt
agaaatcata ttcataccca ttgcgtcacc agtagttgtt 1560ctaaatctca
tgaagagtaa atctcctgct agacaagttt gaatatgttg cagacgtgca
1620aatcttgatg tagagttaaa agctttttta attgcgtttt gtccctcttc
tgagtctaac 1680catatcttac aggcaccaga tcttttcaaa gttgggaaac
ggactactgg gcctcttgtc 1740ataccatcct tagttaaaac agttgttgca
ccaccgccag cattgattgc cttacagcca 1800cgcatggcag aagctaccaa
acaaccctct gtagttgcca ttggtatatg ataagatgta 1860ccatcgataa
ccaaggggcc tataacacca acgggcaaag gcatgtaacc tataacattt
1920tcacaacaag cgccaaatac gcggtcgtag tcataatttt tatatggtaa
acgatcagat 1980gctaatacag gagcttctgc caaaattgaa agagccttcc
tacgtaccgc aaccgctctc 2040gtagtatcac ctaatttttt ctccaaagcg
tacaaaggta acttaccgtg aataaccaag 2100gcagcgacct ctttgttctt
caattgtttt gtatttccac tacttaataa tgcttctaat 2160tcttctaaag
gacgtatttt cttatccaag ctttcaatat cgcgggaatc atcttcctca
2220ctagatgatg aaggtcctga tgagctcgat tgcgcagatg ataaactttt
gactttcgat 2280ccagaaatga ctgttttatt ggttaaaact ggtgtagaag
ccttttgtac aggagcagta 2340aaagacttct tggtgacttc agtcttcacc
aattggtctg cagccattat agttttttct 2400ccttgacgtt aaagtataga
ggtatattaa caattttttg ttgatacttt tatgacattt 2460gaataagaag
taatacaaac cgaaaatgtt gaaagtatta gttaaagtgg ttatgcagct
2520tttgcattta tatatctgtt aatagatcaa aaatcatcgc ttcgctgatt
aattacccca 2580gaaataaggc taaaaaacta atcgcattat tatcctatgg
ttgttaattt gattcgttga 2640tttgaaggtt tgtggggcca ggttactgcc
aatttttcct cttcataacc ataaaagcta 2700gtattgtaga atctttattg
ttcggagcag tgcggcgcga ggcacatctg cgtttcagga 2760acgcgaccgg
tgaagaccag gacgcacgga ggagagtctt ccgtcggagg gctgtcgccc
2820gctcggcggc ttctaatccg tacttcaata tagcaatgag cagttaagcg
tattactgaa 2880agttccaaag agaaggtttt tttaggctaa gataatgggg
ctctttacat ttccacaaca 2940tataagtaag attagatatg gatatgtata
tggtggtatt gccatgtaat atgattatta 3000aacttctttg cgtccatcca
aaaaaaaagt aagaattttt gaaaattcaa tataaatggc 3060ttcagaaaaa
gaaattagga gagagagatt cttgaacgtt ttccctaaat tagtagagga
3120attgaacgca tcgcttttgg cttacggtat gcctaaggaa gcatgtgact
ggtatgccca 3180ctcattgaac tacaacactc caggcggtaa gctaaataga
ggtttgtccg ttgtggacac 3240gtatgctatt ctctccaaca agaccgttga
acaattgggg caagaagaat acgaaaaggt 3300tgccattcta ggttggtgca
ttgagttgtt gcaggcttac ttcttggtcg ccgatgatat 3360gatggacaag
tccattacca gaagaggcca accatgttgg tacaaggttc ctgaagttgg
3420ggaaattgcc atcaatgacg cattcatgtt agaggctgct atctacaagc
ttttgaaatc 3480tcacttcaga aacgaaaaat actacataga tatcaccgaa
ttgttccatg aggtcacctt 3540ccaaaccgaa ttgggccaat tgatggactt
aatcactgca cctgaagaca aagtcgactt 3600gagtaagttc tccctaaaga
agcactcctt catagttact ttcaagactg cttactattc 3660tttctacttg
cctgtcgcat tggccatgta cgttgccggt atcacggatg aaaaggattt
3720gaaacaagcc agagatgtct tgattccatt gggtgaatac ttccaaattc
aagatgacta 3780cttagactgc ttcggtaccc cagaacagat cggtaagatc
ggtacagata tccaagataa 3840caaatgttct tgggtaatca acaaggcatt
ggaacttgct tccgcagaac aaagaaagac 3900tttagacgaa aattacggta
agaaggactc agtcgcagaa gccaaatgca aaaagatttt 3960caatgacttg
aaaattgaac agctatacca cgaatatgaa gagtctattg ccaaggattt
4020gaaggccaaa atttctcagg tcgatgagtc tcgtggcttc aaagctgatg
tcttaactgc 4080gttcttgaac aaagtttaca agagaagcaa atagaactaa
cgctaatcga taaaacatta 4140gatttcaaac tagataagga ccatgtataa
gaactatata cttccaatat aatatagtat 4200aagctttaag atagtatctc
tcgatctacc gttccacgtg actagtccaa ggattttttt 4260taacccggga
tatatgtgta ctttgcagtt atgacgccag atggcagtag tggaagatat
4320tctttattga aaaatagctt gtcaccttac gtacaatctt gatccggagc
ttttcttttt 4380ttgccgatta agaattcggt cgaaaaaaga aaaggagagg
gccaagaggg agggcattgg 4440tgactattga gcacgtgagt atacgtgatt
aagcacacaa aggcagcttg gagtatgtct 4500gttattaatt tcacaggtag
ttctggtcca ttggtgaaag tttgcggctt gcagagcaca 4560gaggccgcag
aatgtgctct agattccgat gctgacttgc tgggtattat atgtgtgccc
4620aatagaaaga gaacaattga cccggttatt gcaaggaaaa tttcaagtct
tgtaaaagca 4680tataaaaata gttcaggcac tccgaaatac ttggttggcg
tgtttcgtaa tcaacctaag 4740gaggatgttt tggctctggt caatgattac
ggcattgata tcgtccaact gcatggagat 4800gagtcgtggc aagaatacca
agagttcctc ggtttgccag ttattaaaag actcgtattt 4860ccaaaagact
gcaacatact actcagtgca gcttcacaga aacctcattc gtttattccc
4920ttgtttgatt cagaagcagg tgggacaggt gaacttttgg attggaactc
gatttctgac 4980tgggttggaa ggcaagagag ccccgaaagc ttacatttta
tgttagctgg tggactgacg 5040ccgtttaaac 5050516081DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
51gtttaaactt ttccaatagg tggttagcaa tcgtcttact ttctaacttt tcttaccttt
60tacatttcag caatatatat atatatattt caaggatata ccattctaat gtctgcccct
120aagaagatcg tcgttttgcc aggtgaccac gttggtcaag aaatcacagc
cgaagccatt 180aaggttctta aagctatttc tgatgttcgt tccaatgtca
agttcgattt cgaaaatcat 240ttaattggtg gtgctgctat cgatgctaca
ggtgttccac ttccagatga ggcgctggaa 300gcctccaaga aggctgatgc
cgttttgtta ggtgctgtgg gtggtcctaa atggggtacc 360ggtagtgtta
gacctgaaca aggtttacta aaaatccgta aagaacttca attgtacgcc
420aacttaagac catgtaactt tgcatccgac tctcttttag acttatctcc
aatcaagcca 480caatttgcta aaggtactga cttcgttgtt gtcagagaat
tagtgggagg tatttacttt 540ggtaagagaa aggaagacgt ttagcttgcc
tcgtccccgc cgggtcaccc ggccagcgac 600atggaggccc agaataccct
ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 660gactgtcgcc
cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat
720tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac
ctgcgagcag 780ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg
ccgcgcccct gtagagaaat 840ataaaaggtt aggatttgcc actgaggttc
ttctttcata tacttccttt taaaatcttg 900ctaggataca gttctcacat
cacatccgaa cataaacaac catggcagaa ccagcccaaa 960aaaagcaaaa
acaaactgtt caggagcgca aggcgtttat ctcccgtatc actaatgaaa
1020ctaaaattca aatcgctatt tcgctgaatg gtggttatat tcaaataaaa
gattcgattc 1080ttcctgcaaa gaaggatgac gatgtagctt cccaagctac
tcagtcacag gtcatcgata 1140ttcacacagg tgttggcttt ttggatcata
tgatccatgc gttggcaaaa cactctggtt 1200ggtctcttat tgttgaatgt
attggtgacc tgcacattga cgatcaccat actaccgaag 1260attgcggtat
cgcattaggg caagcgttca aagaagcaat gggtgctgtc cgtggtgtaa
1320aaagattcgg tactgggttc gcaccattgg atgaggcgct atcacgtgcc
gtagtcgatt 1380tatctagtag accatttgct gtaatcgacc ttggattgaa
gagagagatg attggtgatt 1440tatccactga aatgattcca cactttttgg
aaagtttcgc ggaggcggcc agaattactt 1500tgcatgttga ttgtctgaga
ggtttcaacg atcaccacag aagtgagagt gcgttcaagg 1560ctttggctgt
tgccataaga gaagctattt ctagcaatgg caccaatgac gttccctcaa
1620ccaaaggtgt tttgatgtga agtactgaca ataaaaagat tcttgttttc
aagaacttgt 1680catttgtata gtttttttat attgtagttg ttctatttta
atcaaatgtt agcgtgattt 1740atattttttt tcgcctcgac atcatctgcc
cagatgcgaa gttaagtgcg cagaaagtaa 1800tatcatgcgt caatcgtatg
tgaatgctgg tcgctatact gctgtcgatt cgatactaac 1860gccgccatcc
acccgggttt ctcattcaag tggtaactgc tgttaaaatt aagatattta
1920taaattgaag cttggtcgtt ccgaccaata ccgtagggaa acgtaaatta
gctattgtaa 1980aaaaaggaaa agaaaagaaa agaaaaatgt tacatatcga
attgatctta ttcctttggt 2040agaccagtct ttgcgtcaat caaagattcg
tttgtttctt gtgggcctga accgacttga 2100gttaaaatca ctctggcaac
atccttttgc aactcaagat ccaattcacg tgcagtaaag 2160ttagatgatt
caaattgatg gttgaaagcc tcaagctgct cagtagtaaa tttcttgtcc
2220catccaggaa cagagccaaa caatttatag ataaatgcaa agagtttcga
ctcattttca 2280gctaagtagt acaacacagc atttggacct gcatcaaacg
tgtatgcaac gattgtttct 2340ccgtaaaact gattaatggt gtggcaccaa
ctgatgatac gcttggaagt gtcattcatg 2400tagaatattg gagggaaaga
gtccaaacat gtggcatgga aagagttgga atccatcatt 2460gtttcctttg
caaaggtggc gaaatctttt tcaacaatgg ctttacgcat gacttcaaat
2520ctctttggta cgacatgttc aattctttct ttaaatagtt cggaggttgc
cacggtcaat 2580tgcataccct gagtggaact cacatccttt ttaatatcgc
tgacaactag gacacaagct 2640ttcatctgag gccagtcaga gctgtctgcg
atttgtactg ccatggaatc atgaccatct 2700tcagcttttc ccatttccca
ggccacgtat ccgccaaaca acgatctaca agctgaacca 2760gacccctttc
ttgctattct agatatttct gaagttgact gtggtaattg gtataactta
2820gcaattgcag agaccaatgc agcaaagcca gcagcggagg aagctaaacc
agctgctgta 2880ggaaagttat tttcggagac aatgtggagt ttccattgag
ataatgtggg caatgaggcg 2940tccttcgatt ccatttcctt tcttaattgg
cgtaggtcgc gcagacaatt ttgagttctt 3000tcattgtcga tgctgtgtgg
ttctccattt aaccacaaag tgtcgcgttc aaactcaggt 3060gcagtagccg
cagaggtcaa cgttctgagg tcatcttgcg ataaagtcac tgatatggac
3120gaattggtgg gcagattcaa cttcgtgtcc cttttccccc aatacttaag
ggttgcgatg 3180ttgacgggtg cggtaacgga tgctgtgtaa acggtcatta
tagttttttc tccttgacgt 3240taaagtatag aggtatatta acaatttttt
gttgatactt ttatgacatt tgaataagaa 3300gtaatacaaa ccgaaaatgt
tgaaagtatt agttaaagtg gttatgcagc ttttgcattt 3360atatatctgt
taatagatca aaaatcatcg cttcgctgat taattacccc agaaataagg
3420ctaaaaaact aatcgcatta ttatcctatg gttgttaatt tgattcgttg
atttgaaggt 3480ttgtggggcc aggttactgc caatttttcc tcttcataac
cataaaagct agtattgtag 3540aatctttatt gttcggagca gtgcggcgcg
aggcacatct gcgtttcagg aacgcgaccg 3600gtgaagacca ggacgcacgg
aggagagtct tccgtcggag ggctgtcgcc cgctcggcgg 3660cttctaatcc
gtacttcaat atagcaatga gcagttaagc gtattactga aagttccaaa
3720gagaaggttt ttttaggcta agataatggg gctctttaca tttccacaac
atataagtaa 3780gattagatat ggatatgtat atggtggtat tgccatgtaa
tatgattatt aaacttcttt 3840gcgtccatcc aaaaaaaaag taagaatttt
tgaaaattca atataaatgt cagagttgag 3900agccttcagt gccccaggga
aagcgttact agctggtgga tatttagttt tagatccgaa 3960atatgaagca
tttgtagtcg gattatcggc aagaatgcat gctgtagccc atccttacgg
4020ttcattgcaa gagtctgata agtttgaagt gcgtgtgaaa agtaaacaat
ttaaagatgg 4080ggagtggctg taccatataa gtcctaaaac tggcttcatt
cctgtttcga taggcggatc 4140taagaaccct ttcattgaaa aagttatcgc
taacgtattt agctacttta agcctaacat 4200ggacgactac tgcaatagaa
acttgttcgt tattgatatt ttctctgatg atgcctacca 4260ttctcaggag
gacagcgtta ccgaacatcg tggcaacaga agattgagtt ttcattcgca
4320cagaattgaa gaagttccca aaacagggct gggctcctcg gcaggtttag
tcacagtttt 4380aactacagct ttggcctcct tttttgtatc ggacctggaa
aataatgtag acaaatatag 4440agaagttatt cataatttat cacaagttgc
tcattgtcaa gctcagggta aaattggaag 4500cgggtttgat gtagcggcgg
cagcatatgg atctatcaga tatagaagat tcccacccgc 4560attaatctct
aatttgccag atattggaag tgctacttac ggcagtaaac tggcgcattt
4620ggttaatgaa gaagactgga atataacgat taaaagtaac catttacctt
cgggattaac 4680tttatggatg ggcgatatta agaatggttc agaaacagta
aaactggtcc agaaggtaaa 4740aaattggtat gattcgcata tgccggaaag
cttgaaaata tatacagaac tcgatcatgc 4800aaattctaga tttatggatg
gactatctaa actagatcgc ttacacgaga ctcatgacga 4860ttacagcgat
cagatatttg agtctcttga gaggaatgac tgtacctgtc aaaagtatcc
4920tgagatcaca gaagttagag atgcagttgc cacaattaga cgttccttta
gaaaaataac 4980taaagaatct ggtgccgata tcgaacctcc cgtacaaact
agcttattgg atgattgcca 5040gaccttaaaa ggagttctta cttgcttaat
acctggtgct ggtggttatg acgccattgc 5100agtgattgct aagcaagatg
ttgatcttag ggctcaaacc gctgatgaca aaagattttc 5160taaggttcaa
tggctggatg taactcaggc tgactggggt gttaggaaag aaaaagatcc
5220ggaaacttat cttgataaat aacttaaggt agataatagt ggtccatgtg
acatctttat 5280aaatgtgaag tttgaagtga ccgcgcttaa catctaacca
ttcatcttcc gatagtactt 5340gaaattgttc ctttcggcgg catgataaaa
ttcttttaat gggtacaagc tacccgggaa 5400agattctctt tttttatgat
atttgtacat aaactttata aatgaaattc ataatagaaa 5460cgacacgaaa
ttacaaaatg gaatatgttc atagggtaga cgaaactata tacgcaatct
5520acatacattt atcaagaagg agaaaaagga ggatgtaaag gaatacaggt
aagcaaattg 5580atactaatgg ctcaacgtga taaggaaaaa gaattgcact
ttaacattaa tattgacaag 5640gaggagggca ccacacaaaa agttaggtgt
aacagaaaat catgaaacta tgattcctaa 5700tttatatatt ggaggatttt
ctctaaaaaa aaaaaaatac aacaaataaa aaacactcaa 5760tgacctgacc
atttgatgga gtttaagtca ataccttctt gaaccatttc ccataatggt
5820gaaagttccc tcaagaattt tactctgtca gaaacggcct taacgacgta
gtcgacctcc 5880tcttcagtac taaatctacc aataccaaat ctgatggaag
aatgggctaa tgcatcatcc 5940ttacccagcg catgtaaaac ataagaaggt
tctagggaag cagatgtaca ggctgaaccc 6000gaggataatg cgatatccct
tagtgccatc aataaagatt ctccttccac gtaggcgaaa 6060gaaacgttaa
cacgtttaaa c 6081524933DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 52gtttaaacta
ctcagtatat taagtttcga attgaagggc gaactcttat tcgaagtcgg 60agtcaccaca
acacttccgc ccatactctc cgaatcctcg tttcctaaag taagtttact
120tccacttgta ggcctattat taatgatatc tgaataatcc tctattaggg
ttggatcatt 180cagtagcgcg tgcgattgaa aggagtccat gcccgacgtc
gacgtgatta gcgaaggcgc 240gtaaccattg tcatgtctag cagctataga
actaacctcc ttgacaccac ttgcggaagt 300ctcatcaaca tgctcttcct
tattactcat tctcttacca agcagagaat gttatctaaa 360aactacgtgt
atttcacctc tttctcgact tgaacacgtc caactcctta agtactacca
420cagccaggaa agaatggatc cagttctaca cgatagcaaa gcagaaaaca
caaccagcgt 480acccctgtag aagcttcttt gtttacagca cttgatccat
gtagccatac tcgaaatttc 540aactcatctg aaacttttcc tgaaggttga
aaaagaatgc cataagggtc acccgaagct 600tattcacgcc cgggagttat
gacaattaca acaacagaat tctttctata tatgcacgaa 660cttgtaatat
ggaagaaatt atgacgtaca aactataaag taaatatttt acgtaacaca
720tggtgctgtt gtgcttcttt ttcaagagaa taccaatgac gtatgactaa
gtttaggatt 780taatgcaggt gacggaccca tctttcaaac gatttatatc
agtggcgtcc aaattgttag 840gttttgttgg ttcagcaggt ttcctgttgt
gggtcatatg actttgaacc aaatggccgg 900ctgctagggc agcacataag
gataattcac ctgccaagac ggcacaggca actattcttg 960ctaattgacg
tgcgttggta ccaggagcgg tagcatgtgg gcctcttaca cctaataagt
1020ccaacatggc accttgtggt tctagaacag taccaccacc gatggtacct
acttcgatgg 1080atggcatgga tacggaaatt ctcaaatcac cgtccacttc
tttcatcaat gttatacagt 1140tggaactttc gacattttgt gcaggatctt
gtcctaatgc caagaaaaca gctgtcacta 1200aattagctgc atgtgcgtta
aatccaccaa cagacccagc cattgcagat ccaaccaaat 1260tcttagcaat
gttcaactca accaatgcgg aaacatcact ttttaacact tttctgacaa
1320catcaccagg aatagtagct tctgcgacga cactcttacc acgaccttcg
atccagttga 1380tggcagctgg ttttttgtcg gtacagtagt taccagaaac
ggagacaacc tccatatctt 1440cccagccata ctcttctacc atttgcttta
atgagtattc gacaccctta gaaatcatat 1500tcatacccat tgcgtcacca
gtagttgttc taaatctcat gaagagtaaa tctcctgcta 1560gacaagtttg
aatatgttgc agacgtgcaa atcttgatgt agagttaaaa gcttttttaa
1620ttgcgttttg tccctcttct gagtctaacc atatcttaca ggcaccagat
cttttcaaag 1680ttgggaaacg gactactggg cctcttgtca taccatcctt
agttaaaaca gttgttgcac 1740caccgccagc attgattgcc ttacagccac
gcatggcaga agctaccaaa caaccctctg 1800tagttgccat tggtatatga
taagatgtac catcgataac caaggggcct ataacaccaa 1860cgggcaaagg
catgtaacct ataacatttt cacaacaagc gccaaatacg cggtcgtagt
1920cataattttt atatggtaaa cgatcagatg ctaatacagg agcttctgcc
aaaattgaaa 1980gagccttcct acgtaccgca accgctctcg tagtatcacc
taattttttc tccaaagcgt 2040acaaaggtaa cttaccgtga ataaccaagg
cagcgacctc tttgttcttc aattgttttg 2100tatttccact acttaataat
gcttctaatt cttctaaagg acgtattttc ttatccaagc 2160tttcaatatc
gcgggaatca tcttcctcac tagatgatga aggtcctgat gagctcgatt
2220gcgcagatga taaacttttg actttcgatc cagaaatgac tgttttattg
gttaaaactg 2280gtgtagaagc cttttgtaca ggagcagtaa aagacttctt
ggtgacttca gtcttcacca 2340attggtctgc agccattata gttttttctc
cttgacgtta aagtatagag gtatattaac 2400aattttttgt tgatactttt
atgacatttg aataagaagt aatacaaacc gaaaatgttg 2460aaagtattag
ttaaagtggt tatgcagctt ttgcatttat atatctgtta atagatcaaa
2520aatcatcgct tcgctgatta attaccccag aaataaggct aaaaaactaa
tcgcattatt 2580atcctatggt tgttaatttg attcgttgat ttgaaggttt
gtggggccag gttactgcca 2640atttttcctc ttcataacca taaaagctag
tattgtagaa tctttattgt tcggagcagt 2700gcggcgcgag gcacatctgc
gtttcaggaa cgcgaccggt gaagaccagg acgcacggag 2760gagagtcttc
cgtcggaggg ctgtcgcccg ctcggcggct tctaatccgt acttcaatat
2820agcaatgagc agttaagcgt attactgaaa gttccaaaga gaaggttttt
ttaggctaag 2880ataatggggc tctttacatt tccacaacat ataagtaaga
ttagatatgg atatgtatat 2940ggtggtattg ccatgtaata tgattattaa
acttctttgc gtccatccaa aaaaaaagta 3000agaatttttg aaaattcaat
ataaatgact gccgacaaca atagtatgcc ccatggtgca 3060gtatctagtt
acgccaaatt agtgcaaaac caaacacctg aagacatttt ggaagagttt
3120cctgaaatta ttccattaca acaaagacct aatacccgat ctagtgagac
gtcaaatgac 3180gaaagcggag aaacatgttt ttctggtcat gatgaggagc
aaattaagtt aatgaatgaa 3240aattgtattg ttttggattg ggacgataat
gctattggtg ccggtaccaa gaaagtttgt 3300catttaatgg aaaatattga
aaagggttta ctacatcgtg cattctccgt ctttattttc 3360aatgaacaag
gtgaattact tttacaacaa agagccactg aaaaaataac tttccctgat
3420ctttggacta acacatgctg ctctcatcca ctatgtattg atgacgaatt
aggtttgaag 3480ggtaagctag acgataagat taagggcgct attactgcgg
cggtgagaaa actagatcat 3540gaattaggta ttccagaaga tgaaactaag
acaaggggta agtttcactt tttaaacaga 3600atccattaca tggcaccaag
caatgaacca tggggtgaac atgaaattga ttacatccta 3660ttttataaga
tcaacgctaa agaaaacttg actgtcaacc caaacgtcaa tgaagttaga
3720gacttcaaat gggtttcacc aaatgatttg aaaactatgt ttgctgaccc
aagttacaag 3780tttacgcctt ggtttaagat tatttgcgag aattacttat
tcaactggtg ggagcaatta 3840gatgaccttt ctgaagtgga aaatgacagg
caaattcata gaatgctata acaacgcgtc 3900aataatatag gctacataaa
aatcataata actttgttat catagcaaaa tgtgatataa 3960aacgtttcat
ttcacctgaa aaatagtaaa aataggcgac aaaaatcctt agtaatatgt
4020aaactttatt ttctttattt acccgggagt cagtctgact cttgcgagag
atgaggatgt 4080aataatacta atctcgaaga tgccatctaa tacatataga
catacatata tatatatata 4140cattctatat attcttaccc agattctttg
aggtaagacg gttgggtttt atcttttgca 4200gttggtacta ttaagaacaa
tcgaatcata agcattgctt acaaagaata cacatacgaa 4260atattaacga
taatgtcaat tacgaagact gaactggacg gtatattgcc attggtggcc
4320agaggtaaag ttagagacat atatgaggta gacgctggta cgttgctgtt
tgttgctacg 4380gatcgtatct ctgcatatga cgttattatg gaaaacagca
ttcctgaaaa ggggatccta 4440ttgaccaaac tgtcagagtt ctggttcaag
ttcctgtcca acgatgttcg taatcatttg 4500gtcgacatcg ccccaggtaa
gactattttc gattatctac ctgcaaaatt gagcgaacca 4560aagtacaaaa
cgcaactaga agaccgctct ctattggttc acaaacataa actaattcca
4620ttggaagtaa ttgtcagagg ctacatcacc ggatctgctt ggaaagagta
cgtaaaaaca 4680ggtactgtgc atggtttgaa acaacctcaa ggacttaaag
aatctcaaga gttcccagaa 4740ccaatcttca ccccatcgac caaggctgaa
caaggtgaac atgacgaaaa catctctcct 4800gcccaggccg ctgagctggt
gggtgaagat ttgtcacgta gagtggcaga actggctgta 4860aaactgtact
ccaagtgcaa agattatgct aaggagaagg gcatcatcat cgcagacact
4920aaattgttta aac 4933538425DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 53tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatcga ctacgtcgta aggccgtttc tgacagagta
aaattcttga gggaactttc 240accattatgg gaaatgcttc aagaaggtat
tgacttaaac tccatcaaat ggtcaggtca 300ttgagtgttt tttatttgtt
gtattttttt ttttttagag aaaatcctcc aatatcaaat 360taggaatcgt
agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
420ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc
cattagtatc 480aatttgctta cctgtattcc tttactatcc tcctttttct
ccttcttgat aaatgtatgt 540agattgcgta tatagtttcg tctaccctat
gaacatattc cattttgtaa tttcgtgtcg 600tttctattat gaatttcatt
tataaagttt atgtacaaat atcataaaaa aagagaatct 660ttttaagcaa
ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
720ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt
aactgcatct 780tcaatggcct taccttcttc aggcaagttc aatgacaatt
tcaacatcat tgcagcagac 840aagatagtgg cgatagggtc aaccttattc
tttggcaaat ctggagcaga accgtggcat 900ggttcgtaca aaccaaatgc
ggtgttcttg tctggcaaag aggccaagga cgcagatggc 960aacaaaccca
aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
1020ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat
catggcggca 1080gaatcaatca attgatgttg aaccttcaat gtagggaatt
cgttcttgat ggtttcctcc 1140acagtttttc tccataatct tgaagaggcc
aaaagattag ctttatccaa ggaccaaata 1200ggcaatggtg gctcatgttg
tagggccatg aaagcggcca ttcttgtgat tctttgcact 1260tctggaacgg
tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
1320ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt
acctttagca 1380aattgtggct tgattggaga taagtctaaa agagagtcgg
atgcaaagtt acatggtctt 1440aagttggcgt acaattgaag ttctttacgg
atttttagta aaccttgttc aggtctaaca 1500ctaccggtac cccatttagg
accagccaca gcacctaaca aaacggcatc aaccttcttg 1560gaggcttcca
gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
1620attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat
agctttaaga 1680accttaatgg cttcggctgt gatttcttga ccaacgtggt
cacctggcaa aacgacgatc 1740ttcttagggg cagacattac aatggtatat
ccttgaaata tatataaaaa aaggcgcctt 1800agaccgctcg gccaaacaac
caattacttg ttgagaaata gagtataatt atcctataaa 1860tataacgttt
ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat
1920gtggattttg atgtaattgt tgggattcca tttttaataa ggcaataata
ttaggtatgt 1980ggatatacta gaagttctcc tcgaccgtcg atatgcggtg
tgaaataccg cacagatgcg 2040taaggagaaa ataccgcatc aggaaattgt
aaacgttaat attttgttaa aattcgcgtt 2100aaatttttgt taaatcagct
cattttttaa ccaataggcc gaaatcggca aaatccctta 2160taaatcaaaa
gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
2220actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
agggcgatgg 2280cccactacgt ggaagatccg aggcctagct ttaacgaacg
cagaattttc gagttattaa 2340acttaaaata cgctgaaccc gaacatagaa
atatcgaatg ggaaaaaaaa actgcataaa 2400ggcattaaaa gaggagcgaa
ttttttttta ataaaaatct taataatcat taaaagataa 2460ataatagtct
atatatacgt atataaataa aaaatattca aaaaataaaa taaactatta
2520ttttagcgta aaggatgggg aaagagaaaa gaaaaaaatt gatctatcga
tttcaattca 2580attcaattta tttcttttcg gataagaaag caacacctgg
caattcctta ccttccaata 2640attccaaaga agcaccacca ccagtagaga
catgggagac ccgggccatg gttagataga 2700catagggtaa actagcaatg
atttgatcaa atgcttgtat tcatctccca ttctcgtaaa 2760attgtcttta
cctgcatatt ggacctctaa aaattggcaa agatatataa cagccataag
2820taaaggtctt gggatattct ttgttgttaa atactctctg tttatgtctt
tccaaacgtc 2880ctccacttcc ttataaatca gtgtctgagc atattcttcg
ttgacattgt attccttcat 2940gtaagattct aaagagcttg aactatgttt
tctctcctgt tccgctttat gagtcatcag 3000gtcatttaat ctcctaccca
gaataccact gtaacggaat aaaggcggag cagatacagc 3060ccactcaact
gattccttag tgaaaatatc gctcattcct agataacagg tagttgttag
3120caagtttgca ccaccagtga taataactac gggatcgtgc tcttcagttg
tcggtatgtg 3180tccttcatta gcccatttcg cttctaccat tagattcctt
acgaattctt taacgaactc 3240cttcccacag ttgaataaat cagttctacc
ttctttggcc agaaactcct ccatttctgt 3300gtaggtatcc atgaataatt
tgtaaatagg cttcatgtat tccggcaacg tgtctaagca 3360ggtgatcgac
catctttcca cggcttcagt gaaaatcttt aactcctcgt aagttccata
3420tgcgtcatac gtgtcatcaa taagtgttat cacagcaact gccttagtga
aaaaaactct 3480agctcttgaa tactggggtt cgtaaccaga acctaaaccc
caaaaatagc attcaacgat 3540acgatctctc agacatgggg catttttctt
aatatcaaat gccttccacc acttgcatac 3600gtgactcaac tcttccttat
gtaggctctg caatagattg aactccagtt tagctaactt 3660tagcagagtt
ttattatggg agtcttgttg ctgatagaag ggtatgtact gggcggcctc
3720gatccttggc aatctcttcc acaatggttg ctttaaagct ctctggattt
cagtgaataa 3780agcggggttt gtactaaacg cgtcctttgt cataatcgat
agccttgatc ttgtgaatcc 3840cagggcatct tcaagaatta tttcgcccgg
aactctcatg gacgtagcct catataattc 3900caacaatcct tcaacatcat
tcgctaacga ttgtttaaaa gcaccattct tgtctttata 3960gttattaaac
acatcacacg tgacatagta tccttgttta cgcatcagcc taaaccataa
4020gctagacctg tcgccattcc aattatcacc ataggtctcg taaatacatt
gcaatgcatg 4080atcaatttca cgttcaaaat gatacggaat acctaaacgt
tgaatctcgt caatcagctt 4140caacaaattt gcatgtttca taggaatatc
caatgcttcc tttaacaact gtcttacttc 4200cttctttaga tcgtttacta
tttgctccac accctgttca acttgtttct cataaatcaa 4260aaattgatcg
ccccaaatag aaggtgggaa atttgcaatt ggccttatag gtttctcttc
4320agtcaaggcc attgttttct gcagatccgg ggttttttct ccttgacgtt
aaagtataga 4380ggtatattaa caattttttg ttgatacttt tattacattt
gaataagaag taatacaaac 4440cgaaaatgtt gaaagtatta gttaaagtgg
ttatgcagtt tttgcattta tatatctgtt 4500aatagatcaa aaatcatcgc
ttcgctgatt aattacccca gaaataaggc taaaaaacta 4560atcgcattat
catcctatgg ttgttaattt gattcgttca tttgaaggtt tgtggggcca
4620ggttactgcc aatttttcct cttcataacc ataaaagcta gtattgtaga
atctttattg 4680ttcggagcag tgcggcgcga ggcacatctg cgtttcagga
acgcgaccgg tgaagacgag 4740gacgcacgga ggagagtctt ccttcggagg
gctgtcaccc gctcggcggc ttctaatccg 4800tactaagatc tgctttaatt
tggccggcga acgtggcgag aaaggaaggg aagaaagcga 4860aaggagcggg
cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
4920ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc
aggctgcgca 4980actgttggga agggcgatcg gtgcgggcct cttcgctatt
acgccagctg cattaatgaa 5040tcggccaacg cgcggggaga ggcggtttgc
gtattgggcg ctcttccgct tcctcgctca 5100ctgactcgct gcgctcggtc
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 5160taatacggtt
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
5220agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
aggctccgcc 5280cccctgacga gcatcacaaa aatcgacgct caagtcagag
gtggcgaaac ccgacaggac 5340tataaagata ccaggcgttt ccccctggaa
gctccctcgt gcgctctcct gttccgaccc 5400tgccgcttac cggatacctg
tccgcctttc tcccttcggg aagcgtggcg ctttctcata 5460gctcacgctg
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
5520acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
cttgagtcca 5580acccggtaag acacgactta tcgccactgg cagcagccac
tggtaacagg attagcagag 5640cgaggtatgt aggcggtgct acagagttct
tgaagtggtg gcctaactac ggctacacta 5700gaaggacagt atttggtatc
tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5760gtagctcttg
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
5820agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
tctacggggt 5880ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga ttatcaaaaa 5940ggatcttcac ctagatcctt ttaaattaaa
aatgaagttt taaatcaatc taaagtatat 6000atgagtaaac ttggtctgac
agttaccaat gcttaatcag tgaggcacct atctcagcga 6060tctgtctatt
tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
6120gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
cgctcaccgg 6180ctccagattt atcagcaata aaccagccag ccggaagggc
cgagcgcaga agtggtcctg 6240caactttatc cgcctccatc cagtctatta
attgttgccg ggaagctaga gtaagtagtt 6300cgccagttaa tagtttgcgc
aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 6360cgtcgtttgg
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
6420cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
gtcagaagta 6480agttggccgc agtgttatca ctcatggtta tggcagcact
gcataattct cttactgtca 6540tgccatccgt aagatgcttt tctgtgactg
gtgagtactc aaccaagtca ttctgagaat 6600agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat acgggataat accgcgccac 6660atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
6720ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
aactgatctt 6780cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
aacaggaagg caaaatgccg 6840caaaaaaggg aataagggcg acacggaaat
gttgaatact catactcttc ctttttcaat 6900attattgaag catttatcag
ggttattgtc tcatgagcgg atacatattt gaatgtattt 6960agaaaaataa
acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa
7020gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat
ttttcaaaca 7080aagaatctga gctgcatttt tacagaacag aaatgcaacg
cgaaagcgct attttaccaa 7140cgaagaatct gtgcttcatt tttgtaaaac
aaaaatgcaa cgcgagagcg ctaatttttc 7200aaacaaagaa tctgagctgc
atttttacag aacagaaatg caacgcgaga gcgctatttt 7260accaacaaag
aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt
7320tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat
aatgcagtct 7380cttgataact ttttgcactg taggtccgtt aaggttagaa
gaaggctact ttggtgtcta 7440ttttctcttc cataaaaaaa gcctgactcc
acttcccgcg tttactgatt actagcgaag 7500ctgcgggtgc attttttcaa
gataaaggca tccccgatta tattctatac cgatgtggat 7560tgcgcatact
ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt
7620atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt
tacattttcg 7680tattgttttc gattcactct atgaatagtt cttactacaa
tttttttgtc taaagagtaa 7740tactagagat aaacataaaa aatgtagagg
tcgagtttag atgcaagttc aaggagcgaa 7800aggtggatgg gtaggttata
tagggatata gcacagagat atatagcaaa gagatacttt 7860tgagcaatgt
ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc
7920gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa
gcgctctgaa 7980gttcctatac tttctagaga ataggaactt cggaatagga
acttcaaagc gtttccgaaa 8040acgagcgctt ccgaaaatgc aacgcgagct
gcgcacatac agctcactgt tcacgtcgca 8100cctatatctg cgtgttgcct
gtatatatat atacatgaga agaacggcat agtgcgtgtt 8160tatgcttaaa
tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc
8220tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc
actacccttt 8280agctgttcta tatgctgcca ctcctcaatt ggattagtct
catccttcaa tgctatcatt 8340tcctttgata ttggatcata ctaagaaacc
attattatca tgacattaac ctataaaaat 8400aggcgtatca cgaggccctt tcgtc
84255413280DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 54tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg
cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatcga
ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
240accattatgg gaaatgcttc aagaaggtat tgacttaaac tccatcaaat
ggtcaggtca 300ttgagtgttt tttatttgtt gtattttttt ttttttagag
aaaatcctcc aatatcaaat 360taggaatcgt agtttcatga ttttctgtta
cacctaactt tttgtgtggt gccctcctcc 420ttgtcaatat taatgttaaa
gtgcaattct ttttccttat cacgttgagc cattagtatc 480aatttgctta
cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
540agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa
tttcgtgtcg 600tttctattat gaatttcatt tataaagttt atgtacaaat
atcataaaaa aagagaatct 660ttttaagcaa ggattttctt aacttcttcg
gcgacagcat caccgacttc ggtggtactg 720ttggaaccac ctaaatcacc
agttctgata cctgcatcca aaaccttttt aactgcatct 780tcaatggcct
taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
840aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga
accgtggcat 900ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag
aggccaagga cgcagatggc 960aacaaaccca aggaacctgg gataacggag
gcttcatcgg agatgatatc accaaacatg 1020ttgctggtga ttataatacc
atttaggtgg gttgggttct taactaggat catggcggca 1080gaatcaatca
attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
1140acagtttttc tccataatct tgaagaggcc aaaagattag ctttatccaa
ggaccaaata 1200ggcaatggtg gctcatgttg tagggccatg aaagcggcca
ttcttgtgat tctttgcact 1260tctggaacgg tgtattgttc actatcccaa
gcgacaccat caccatcgtc ttcctttctc 1320ttaccaaagt aaatacctcc
cactaattct ctgacaacaa cgaagtcagt acctttagca 1380aattgtggct
tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
1440aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc
aggtctaaca 1500ctaccggtac cccatttagg accagccaca gcacctaaca
aaacggcatc aaccttcttg 1560gaggcttcca gcgcctcatc tggaagtggg
acacctgtag catcgatagc agcaccacca 1620attaaatgat tttcgaaatc
gaacttgaca ttggaacgaa catcagaaat agctttaaga 1680accttaatgg
cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
1740ttcttagggg cagacattac aatggtatat ccttgaaata tatataaaaa
aaggcgcctt 1800agaccgctcg gccaaacaac caattacttg ttgagaaata
gagtataatt atcctataaa 1860tataacgttt ttgaacacac atgaacaagg
aagtacagga caattgattt tgaagagaat 1920gtggattttg atgtaattgt
tgggattcca tttttaataa ggcaataata ttaggtatgt 1980ggatatacta
gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg
2040taaggagaaa ataccgcatc aggaaattgt aaacgttaat attttgttaa
aattcgcgtt 2100aaatttttgt taaatcagct cattttttaa ccaataggcc
gaaatcggca aaatccctta 2160taaatcaaaa gaatagaccg agatagggtt
gagtgttgtt ccagtttgga acaagagtcc 2220actattaaag aacgtggact
ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 2280cccactacgt
ggaagatccg aggcctagct ttaacgaacg cagaattttc gagttattaa
2340acttaaaata cgctgaaccc gaacatagaa atatcgaatg ggaaaaaaaa
actgcataaa 2400ggcattaaaa gaggagcgaa ttttttttta ataaaaatct
taataatcat taaaagataa 2460ataatagtct atatatacgt atataaataa
aaaatattca aaaaataaaa taaactatta 2520ttttagcgta aaggatgggg
aaagagaaaa gaaaaaaatt gatctatcga tttcaattca 2580attcaattta
tttcttttcg gataagaaag caacacctgg caattcctta ccttccaata
2640attccaaaga agcaccacca ccagtagaga catgggagac ccgggccatg
gttagataga 2700catagggtaa actagcaatg atttgatcaa atgcttgtat
tcatctccca ttctcgtaaa 2760attgtcttta cctgcatatt ggacctctaa
aaattggcaa agatatataa cagccataag 2820taaaggtctt gggatattct
ttgttgttaa atactctctg tttatgtctt tccaaacgtc 2880ctccacttcc
ttataaatca gtgtctgagc
atattcttcg ttgacattgt attccttcat 2940gtaagattct aaagagcttg
aactatgttt tctctcctgt tccgctttat gagtcatcag 3000gtcatttaat
ctcctaccca gaataccact gtaacggaat aaaggcggag cagatacagc
3060ccactcaact gattccttag tgaaaatatc gctcattcct agataacagg
tagttgttag 3120caagtttgca ccaccagtga taataactac gggatcgtgc
tcttcagttg tcggtatgtg 3180tccttcatta gcccatttcg cttctaccat
tagattcctt acgaattctt taacgaactc 3240cttcccacag ttgaataaat
cagttctacc ttctttggcc agaaactcct ccatttctgt 3300gtaggtatcc
atgaataatt tgtaaatagg cttcatgtat tccggcaacg tgtctaagca
3360ggtgatcgac catctttcca cggcttcagt gaaaatcttt aactcctcgt
aagttccata 3420tgcgtcatac gtgtcatcaa taagtgttat cacagcaact
gccttagtga aaaaaactct 3480agctcttgaa tactggggtt cgtaaccaga
acctaaaccc caaaaatagc attcaacgat 3540acgatctctc agacatgggg
catttttctt aatatcaaat gccttccacc acttgcatac 3600gtgactcaac
tcttccttat gtaggctctg caatagattg aactccagtt tagctaactt
3660tagcagagtt ttattatggg agtcttgttg ctgatagaag ggtatgtact
gggcggcctc 3720gatccttggc aatctcttcc acaatggttg ctttaaagct
ctctggattt cagtgaataa 3780agcggggttt gtactaaacg cgtcctttgt
cataatcgat agccttgatc ttgtgaatcc 3840cagggcatct tcaagaatta
tttcgcccgg aactctcatg gacgtagcct catataattc 3900caacaatcct
tcaacatcat tcgctaacga ttgtttaaaa gcaccattct tgtctttata
3960gttattaaac acatcacacg tgacatagta tccttgttta cgcatcagcc
taaaccataa 4020gctagacctg tcgccattcc aattatcacc ataggtctcg
taaatacatt gcaatgcatg 4080atcaatttca cgttcaaaat gatacggaat
acctaaacgt tgaatctcgt caatcagctt 4140caacaaattt gcatgtttca
taggaatatc caatgcttcc tttaacaact gtcttacttc 4200cttctttaga
tcgtttacta tttgctccac accctgttca acttgtttct cataaatcaa
4260aaattgatcg ccccaaatag aaggtgggaa atttgcaatt ggccttatag
gtttctcttc 4320agtcaaggcc attgttttct gcagatccgg ggttttttct
ccttgacgtt aaagtataga 4380ggtatattaa caattttttg ttgatacttt
tattacattt gaataagaag taatacaaac 4440cgaaaatgtt gaaagtatta
gttaaagtgg ttatgcagtt tttgcattta tatatctgtt 4500aatagatcaa
aaatcatcgc ttcgctgatt aattacccca gaaataaggc taaaaaacta
4560atcgcattat catcctatgg ttgttaattt gattcgttca tttgaaggtt
tgtggggcca 4620ggttactgcc aatttttcct cttcataacc ataaaagcta
gtattgtaga atctttattg 4680ttcggagcag tgcggcgcga ggcacatctg
cgtttcagga acgcgaccgg tgaagacgag 4740gacgcacgga ggagagtctt
ccttcggagg gctgtcaccc gctcggcggc ttctaatccg 4800tactaagatc
tgctttaatt tggccggcga acgtggcgag aaaggaaggg aagaaagcga
4860aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
accaccacac 4920ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca
ttcgccattc aggctgcgca 4980actgttggga agggcgatcg gtgcgggcct
cttcgctatt acgccagctg aattggagcg 5040acctcatgct atacctgaga
aagcaacctg acctacagga aagagttact caagaataag 5100aattttcgtt
ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata
5160acttatttaa taataaaaat cataaatcat aagaaattcg cttatttaga
agtgtcaaca 5220acgtatctac caacgatttg acccttttcc atcttttcgt
aaatttctgg caaggtagac 5280aagccgacaa ccttgattgg agacttgacc
aaacctctgg cgaagaattg ttaattaaga 5340gtcagtcgac ttaaaaacta
gggaccaata gcaattctgt tttacgttgc attgttgcac 5400ctgaactttc
cgtcatgtca atttgatcat atgaaactcc attgggcaac ttccagttga
5460aatgataaag aatgttggct agtggcagtt gaacattggc caaacctaac
gcagcgccag 5520gacacatacg acgtccagcc ccaaatggta aatattcata
ttcggcgccc atcactgttg 5580ccgaagagtt ttcaaatctt tcaggtataa
acgcttctgc atccttccag tattcaggat 5640ctctattgat cgcaaacaca
ttaacgatta atttcgtttt gttagggata ttataaccag 5700ccaagtttac
tggctgacga cattctctag gtagcactaa cggcaagggt gggtgtagtc
5760taagagtctc tttgatgacc atattcaagt aggacaattc ttgtatatct
tcttcatgta 5820ttttttcttt cccattcaag gccttacgta attcagcctg
aaccttttcc attgctttcg 5880gacattttat tagctcgctt atagcccatt
ctatggtaga acttgaagtg tcggtccctg 5940caccgaacat gtccaaaatt
attgctttga tattatccga agtcagagga aactcagcag 6000aatcctttaa
tctaagtaat acatctaata gggtttcgtt ggttttggat gacgtattta
6060cggtatgttc agctaccaaa ttgtcaatta agttatcaat ctttttacgt
aggctagtta 6120atcttgctct cttaccgctc aagtgatgca agaacttttt
agatgggaaa atatcggcaa 6180catcgaaacc gcctgtttgt ctcagtattt
ctttaacaat ttcagtaagt tccttttgat 6240ctttaattcc cttaccaaac
gcagcacggg atagtatagt ggcaattagt ttaaaaacgt 6300tttcacttaa
atttactggt ctaccactac ctgaagcctt tatttcctgg actaaattcc
6360aacattcttc ttccctcaac gattgaaatg acttaacctt ttttacagac
aacaattcaa 6420gagtacaaat cttccttaat tgtctccagt attccccata
tggagcaagg acaacatcag 6480tgttatgata taaaactatt tccccagtta
aagtttcggg tctattagcg aaagtaatat 6540cgtaggttgt aagaatttcc
ttagcccact taggactcga cacgactatt gtgggtacct 6600ctcccaattg
aaggtgcatt agcgaaccat attttctcgc taaatccctt acacccctgt
6660gtggtgtggt tccgatcaaa tggtgcatgt gaccaatgat gggtagcctc
caaggttccg 6720gcaaggactt tttagttgac ttacttctag tggcaaattt
gtacacgaac aacaaaatag 6780ttgctaaagc aattgatgta gttaaagata
gtgccatagc ctttaaaatt gacttcattg 6840ttttcctagg cctttagtga
gggttgaatt cgaattttca aaaattctta cttttttttt 6900ggatggacgc
aaagaagttt aataatcata ttacatggca ttaccaccat atacatatcc
6960atatacatat ccatatctaa tcttacttat atgttgtgga aatgtaaaga
gccccattat 7020cttagcctaa aaaaaccttc tctttggaac tttcagtaat
acgcttaact gctcattgct 7080atattgaagt acggattaga agccgccgag
cgggtgacag ccctccgaag gaagactctc 7140ctccgtgcgt cctcgtcttc
accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc 7200actgctccga
acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat
7260tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca
accataggat 7320gataatgcga ttagtttttt agccttattt ctggggtaat
taatcagcga agcgatgatt 7380tttgatctat taacagatat ataaatgcaa
aaactgcata accactttaa ctaatacttt 7440caacattttc ggtttgtatt
acttcttatt caaatgtaat aaaagtatca acaaaaaatt 7500gttaatatac
ctctatactt taacgtcaag gagaaaaaac cccaagcttc ccgggaaaac
7560aatgcaatcg acaacttccg ttaaactatc acctttcgat cttatgactg
ccttgttaaa 7620tggtaaagtt agtttcgaca cgtccaatac ttccgataca
aatataccac tggcggtttt 7680catggaaaac agggaattgc ttatgatatt
aacaaccagt gtggccgttt taattggttg 7740tgtggttgta ttggtatgga
gaagatcatc aagtgccgct aagaaggccg ccgaatcacc 7800agtcattgtc
gtcccaaaga aagtcactga agatgaggtt gatgacggca gaaagaaagt
7860tactgtattt ttcgggacac aaacggggac tgcggaaggt tttgcgaaag
ctctagttga 7920agaagccaag gcaaggtacg aaaaagcagt attcaaagtt
attgatttag atgactacgc 7980cgcagaagat gatgaatacg aagaaaagct
aaagaaagaa tctttggcat tcttcttttt 8040agctacctat ggtgacggag
aaccaacaga taacgccgct agattctata aatggtttac 8100tgaaggagaa
gaaaaaggtg agtggttaga taagttacaa tacgctgtct ttggattggg
8160aaatcgtcaa tatgaacact tcaataagat tgcaaaagtg gtcgatgaaa
aattagttga 8220gcagggggct aaaaggttag tgcctgtcgg tatgggtgat
gacgatcaat gtatcgaaga 8280tgattttact gcttggaagg aattggtttg
gccagaatta gatcagctat tgagggacga 8340agatgacaca agtgtcgcta
ctccgtacac cgccgctgtt ggcgaatatc gtgttgtttt 8400tcacgataaa
cctgaaactt acgatcaaga tcaattgacc aacggacacg cagttcacga
8460cgcccaacac ccatgcagat cgaacgttgc ggtcaagaaa gaattacaca
gtcccttatc 8520cgataggagt tgtactcatt tagaatttga tatttccaat
actggactat cgtatgaaac 8580tggcgaccat gtcggtgtat atgtggaaaa
cctgtctgaa gttgtagatg aagccgaaaa 8640attgattggg cttcctccac
atacatactt ttctgtgcat acagataatg aagatggtac 8700tccacttggc
ggagcctcgt taccacctcc ctttccacca tgtacactta gaaaagctct
8760tgcatcttat gcagatgtac tttcttcacc aaagaaaagt gcattactag
ctctagccgc 8820ccatgctacc gactctactg aagctgaccg tttgaaattc
tttgcttcac ctgctggcaa 8880agacgagtac gcacagtgga ttgtggcatc
tcacagatca ttgctggaag tgatggaagc 8940cttcccatcg gcaaagccac
cattaggcgt gtttttcgca tctgttgccc cacgtttaca 9000gcctagatac
tattccatat cttctagccc aaaatttgcc cccaatcgta ttcatgtgac
9060gtgtgcgctg gtgtatgaac aaactccatc aggaagggta cataaaggtg
tctgtagtac 9120atggatgaaa aacgcggtgc caatgactga atctcaagat
tgttcgtggg caccaattta 9180tgttcgtact tctaatttta gactacctag
tgaccctaaa gtaccagtga ttatgatcgg 9240gcctgggaca ggactagcgc
cattcagagg tttcttacaa gaaagattgg cccaaaagga 9300agcaggtacg
gaattaggaa ccgcaattct attctttggt tgtcgtaata gaaaagttga
9360ctttatatac gaagatgagt taaacaactt cgttgaaact ggagcgttat
cagaattagt 9420gacagcattc tctagggaag gtgcaacaaa agaatacgtc
caacataaaa tgacccaaaa 9480ggccagcgat atatggaatt tgctgtccga
gggtgcctat ttgtacgttt gtggtgatgc 9540aaagggaatg gctaaagatg
ttcacaggac attgcataca attgttcagg aacaaggttc 9600cttggattcc
tctaaggcag aactttatgt taaaaacctt cagatggctg gtagatattt
9660gcgtgatgtt tggtgagcta gctaagatcc gctctaaccg aaaaggaagg
agttagacaa 9720cctgaagtct aggtccctat ttattttttt atagttatgt
tagtattaag aacgttattt 9780atatttcaaa tttttctttt ttttctgtac
agacgcgtgt acgcatgtaa cattatactg 9840aaaaccttgc ttgagaaggt
tttgggacgc tcgaagatcc agctgcatta atgaatcggc 9900caacgcgcgg
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
9960tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata 10020cggttatcca cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa 10080aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct 10140gacgagcatc acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac aggactataa 10200agataccagg
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
10260cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
tcatagctca 10320cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa 10380ccccccgttc agcccgaccg ctgcgcctta
tccggtaact atcgtcttga gtccaacccg 10440gtaagacacg acttatcgcc
actggcagca gccactggta acaggattag cagagcgagg 10500tatgtaggcg
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
10560acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
agttggtagc 10620tcttgatccg gcaaacaaac caccgctggt agcggtggtt
tttttgtttg caagcagcag 10680attacgcgca gaaaaaaagg atctcaagaa
gatcctttga tcttttctac ggggtctgac 10740gctcagtgga acgaaaactc
acgttaaggg attttggtca tgagattatc aaaaaggatc 10800ttcacctaga
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
10860taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
agcgatctgt 10920ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
agataactac gatacgggag 10980ggcttaccat ctggccccag tgctgcaatg
ataccgcgag acccacgctc accggctcca 11040gatttatcag caataaacca
gccagccgga agggccgagc gcagaagtgg tcctgcaact 11100ttatccgcct
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
11160gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
acgctcgtcg 11220tttggtatgg cttcattcag ctccggttcc caacgatcaa
ggcgagttac atgatccccc 11280atgttgtgca aaaaagcggt tagctccttc
ggtcctccga tcgttgtcag aagtaagttg 11340gccgcagtgt tatcactcat
ggttatggca gcactgcata attctcttac tgtcatgcca 11400tccgtaagat
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
11460atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
gccacatagc 11520agaactttaa aagtgctcat cattggaaaa cgttcttcgg
ggcgaaaact ctcaaggatc 11580ttaccgctgt tgagatccag ttcgatgtaa
cccactcgtg cacccaactg atcttcagca 11640tcttttactt tcaccagcgt
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11700aagggaataa
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
11760tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
tatttagaaa 11820aataaacaaa taggggttcc gcgcacattt ccccgaaaag
tgccacctga acgaagcatc 11880tgtgcttcat tttgtagaac aaaaatgcaa
cgcgagagcg ctaatttttc aaacaaagaa 11940tctgagctgc atttttacag
aacagaaatg caacgcgaaa gcgctatttt accaacgaag 12000aatctgtgct
tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
12060aagaatctga gctgcatttt tacagaacag aaatgcaacg cgagagcgct
attttaccaa 12120caaagaatct atacttcttt tttgttctac aaaaatgcat
cccgagagcg ctatttttct 12180aacaaagcat cttagattac tttttttctc
ctttgtgcgc tctataatgc agtctcttga 12240taactttttg cactgtaggt
ccgttaaggt tagaagaagg ctactttggt gtctattttc 12300tcttccataa
aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg
12360ggtgcatttt ttcaagataa aggcatcccc gattatattc tataccgatg
tggattgcgc 12420atactttgtg aacagaaagt gatagcgttg atgattcttc
attggtcaga aaattatgaa 12480cggtttcttc tattttgtct ctatatacta
cgtataggaa atgtttacat tttcgtattg 12540ttttcgattc actctatgaa
tagttcttac tacaattttt ttgtctaaag agtaatacta 12600gagataaaca
taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg
12660gatgggtagg ttatataggg atatagcaca gagatatata gcaaagagat
acttttgagc 12720aatgtttgtg gaagcggtat tcgcaatatt ttagtagctc
gttacagtcc ggtgcgtttt 12780tggttttttg aaagtgcgtc ttcagagcgc
ttttggtttt caaaagcgct ctgaagttcc 12840tatactttct agagaatagg
aacttcggaa taggaacttc aaagcgtttc cgaaaacgag 12900cgcttccgaa
aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat
12960atctgcgtgt tgcctgtata tatatataca tgagaagaac ggcatagtgc
gtgtttatgc 13020ttaaatgcgt acttatatgc gtctatttat gtaggatgaa
aggtagtcta gtacctcctg 13080tgatattatc ccattccatg cggggtatcg
tatgcttcct tcagcactac cctttagctg 13140ttctatatgc tgccactcct
caattggatt agtctcatcc ttcaatgcta tcatttcctt 13200tgatattgga
tcatactaag aaaccattat tatcatgaca ttaacctata aaaataggcg
13260tatcacgagg ccctttcgtc 13280
* * * * *